Abstract The male rat reproductive system is highly sensitive to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) when exposure occurs during fetal and neonatal development. Our objective was to determine the relative contributions of in utero versus lactational TCDD exposure to effects on male reproductive function. Pregnant Holtzman rats were treated on Day 15 of gestation with TCDD (1.0 μg/kg) or vehicle (control). At birth litters were standardized to five males and five females and fostered to dams of the same treatment or cross-fostered to darns of the opposite treatment. Four treatment groups were assessed: male offspring not exposed to TCDD by either route (control) and male offspring exposed to TCDD in utero (IU), via lactation (L), or in utero and via lactation (IUL). During early postnatal development, two androgen sensitive end points, relative anogenital distance and time to testis descent, were not affected by TCDD. However, end points evaluated later during development were altered. Time to separation of the prepuce from the glans penis (an index of pubertal development) was delayed, plasma testosterone concentrations and accessory sex organ weights were reduced, daily sperm production and epididymal sperm reserves were decreased, and sexual behavior was feminized. Certain responses were only produced by IU exposure whereas other responses only occurred following L exposure. Only IU TCDD exposure delayed pubertal development and decreased daily sperm production, while only L TCDD exposure feminized the sexual behavior of male offspring. For most male reproductive end points both IU and L TCDD exposure produced the same responses. Decreases in plasma testosterone concentrations, reductions in weights, protein, and DNA contents of ventral prostate and seminal vesicles, and decreases in epididymal sperm reserves were caused in young adult rats by either IU or L exposure to TCDD. We conclude that the route and timing of TCDD exposure during fetal and neonatal development of the rat determine the profile of male reproductive effects observed and that all effects in the present study, with the notable exception of feminized sexual behavior, can be caused by low level exposure to TCDD via the IU route alone.