Abstract In this study, we explored the impact on both Sertoli cells and gonocytes of a single, relatively low dose of di-(2-ethylhexyl) phthalate (DEHP; 20–500 mg/kg) administered in vivo to 3-day-old rat pups. In parallel, we assessed the potential for two immediate metabolites of DEHP to produce similar testicular changes and began to explore the possible mechanisms involved. Morphological examination revealed the presence of many abnormally large, multi-nucleated germ cells by 24 h posttreatment with DEHP and with its metabolite, mono-ethylhexyl phthalate (MEHP), but not with another metabolite, 2-ethylhexanol (2-EH; all at 1.28 mmol/kg) or with vehicle alone. These cells persisted through 48 h posttreatment, the longest time point examined in our study. We also assessed the rate of Sertoli cell proliferation in pups at intervals after dosage with either chemical or vehicle by administering bromodeoxy uridine (BrdU) 3 h before euthanasia. By 24 h after treatment with DEHP or MEHP, but not 2-EH or vehicle, the number of BrdU-labeled Sertoli cells was obviously diminished in testicular sections. Quantitation of DEHP-treated pups and controls indicates that a dose–response relationship exists between chemical treatment and labeling index (LI) of Sertoli cells, with a LI at the highest DEHP dose tested that was only 20% of that in controls. In addition, when we examined the time course of the effect of an intermediate dose of DEHP, we found that there the LI of Sertoli cells rebounds by 48 h after dosage, when we found the rate of proliferation in treated pups to be significantly higher than in controls. We also explored the potential mechanism involved in the response to DEHP and found serum levels of FSH to be unaffected by the chemical. In addition, study of cell cycle-related proteins including p27kip1 and cyclins D1, D2, and D3 with Western and Northern analysis indicated that cyclin D2 mRNA is specifically down-regulated by DEHP in a dose-dependent manner, and this decrease is manifest as a small, transient but reproducible reduction in the amount of cyclin D2 protein detectable in samples from treated pups compared to controls. Our findings characterize the changes in neonatal Sertoli cells and gonocytes that follow in vivo to low levels of DEHP and its metabolite, MEHP, as well as providing new information on the underlying mechanism and highlighting the extreme sensitivity of the neonatal testis to injury by this toxicant.