ABSTRACT Chlamydia trachomatis represents a significant health concern world-wide. A substantial burden exists due to the prevalence and the combined health and socioeconomic impact of acute and chronic disease. All Chlamydia species express a type III secretion system (T3SS), and this mechanism likely contributes significantly to overall pathogenesis. Chlamydiae are obligate intracellular parasites that undergo a complex developmental cycle, and this biology has historically impeded rapid progress in understanding pathogenesis. The newly acquired ability to inactivate chlamydial genes via insertion or deletion mutagenesis has greatly facilitated characterization of virulence factors. Unfortunately, significant barriers remain in the application of genetics in elucidating molecular mechanisms of virulence. For example, there is currently no mechanism to generate null strains for gene products that are absolutely essential for chlamydial propagation in cell culture. To this end, we have adapted the fluorescence-reported allelic exchange mutagenesis technique to facilitate creation of conditional null strains in essential genes. Preliminary data describing such a situation for a type III effector gene are presented herein. We propose to validate and further develop our approach while revealing the function of a highly important effector protein. At the end of these studies, we will have established new approaches that will benefit the entire Chlamydia research community and advance the understanding of how T3S manifests during chlamydial infection.