Abstract Long INterspersed Element-1 (LINE-1 or L1) transposable element derived sequences comprise approximately 17% of human genomic DNA. Although the overwhelming majority of LINE-1s have been rendered immobile by mutational processes over evolutionary time, an average human genome contains approximately 100 active LINE-1s that can mobilize to new genomic locations via an RNA intermediate by a process known as retrotransposition. The retrotransposition of active LINE-1s in the germline, during early development, or in select somatic cells continues to generate inter- and intra-individual genetic variation and can lead to sporadic cases of human genetic diseases, including hemophilia A, hemophilia B, Duchenne muscular dystrophy, and certain cancers. We posit that a mechanistic understanding of LINE-1 biology is essential to understand the forces that contribute to human disease, human genetic variation, and human evolution. This proposal represents the third competing renewal application of GM060518, and seeks ongoing support to continue our studies of LINE-1 biology. We will build upon our experience and knowledge in the field of mobile genetic elements, use insights gained from studies of the mobility mechanisms of evolutionarily related retrotransposons, and employ a LINE- 1 cultured cell retrotransposition assay in conjunction with an integrated strategy that combines genetic, molecular biological, biochemical, multi-omic, and computational approaches to answer cutting-edge questions in LINE-1 biology. This proposal comprises two Aims, which build upon our extensive toolbox of reagents, previous findings, and preliminary data. In Aim 1, we will elucidate mechanistic aspects of LINE-1 retrotransposition. In Aim 2, we will determine how host proteins act to facilitate or restrict LINE-1 retrotransposition. This project will benefit from a long-standing successful collaboration between the Moran and Kidd laboratories at the University of Michigan and our outstanding research teams. The successful completion of the above Specific Aims will increase our understanding how LINE-1s mobilize to new genomic locations, how the host has evolved mechanisms to prevent unabated LINE-1 retrotransposition, and how LINE-1 has exploited cellular proteins to aid in its retrotransposition. This knowledge also will provide insights into how LINE-1 retrotransposition contributes to human disease, human genetic variation, and human genome evolution.