The 4th chromosome is the final frontier for genetic analysis in Drosophila. Small and devoid of recombination the 4th has long been ignored. Nevertheless, it contains 79 protein coding genes. 92% of the protein coding genes have human counterparts and 68% of these human genes have a disease association. We propose to renew our project to build a Comprehensive Resource for the Drosophila 4th Chromosome. The project is highly visible in the community. It is known as the Fourth Chromosome Resource Project (FCRP) at the Bloomington and Kyoto stock centers as well as in the FlyBase and FlyPush databases. After three years of funding, the FCRP has 446 stocks publicly available and is on track to exceed the proposed goal by 15%. To date, four papers are published and 311 stocks were ordered from Bloomington in 2022. Evidence of impact is that FCRP stocks constitute 0.3% of the Bloomington stock center, but FCRP shipments supported 13% of the NIH grants funding stock orders from Bloomington in 2022. Further evidence of impact is that FCRP stocks for the five most popular 4th genes were ordered at a rate matching the top 3% of all Bloomington stocks. Three of the stock sets have gone well: 1) the UAS.fly cDNA stocks, 2) the UAS.human homolog cDNA stocks and 3) the conversion of MiMIC transgenes to gene trap and protein trap stocks. However, during the project we noted several unmet needs. We propose to address these by continuing to build the two sets that remain incomplete, adding a UAS.RNAi set, plus conducting validation and phenotyping studies on all FCRP stocks. The Specific Aim remains to generate a comprehensive resource enabling modern genetic analysis for protein coding genes on the Drosophila 4th chromosome. One set that will continue is to mutagenize every 4th gene individually on our FRT101F chromosome for loss of function studies and marked single cell clones. The second continuing set is UAS.human cDNA stocks for the top two human relatives of each conserved 4th gene. The need to add a set of UAS.RNAi stocks for genes on the 4th was noted by the PI of the fly Transgenic RNAi Project. Their set of 4th UAS.RNAi stocks is incomplete and the FCRP was encouraged to finish the job. The need for confidence in our stocks will be met with experimental validation. The need to provide investigators with a starting point for genetic analyses will be met by phenotyping. Our systematic observations of phenotypes such as mutant lethality or sterility plus transcript expression and protein localization are the first data for the 58% of 4th genes that were never studied before. Validation of gene trap null mutants and their UAS.fly cDNAs in rescue experiments sets the stage for humanized stocks. When null mutations are rescued by UAS.human cDNAs subsequent mating of rescued flies creates a humanized stock. These stocks provide opportunities to apply Drosophila genetics to the analysis of human gene interactions and function. Taken together, FCRP stocks will...