We showed previously that the chlorotriazine herbicide, atrazine (ATR), delays the onset of pubertal development in female rats. ATR and its biotransformation by-products are present in soil and groundwater. Since current maximum contaminant levels are set only for ATR, it is important to determine whether these by-products can also alter pubertal development and possibly pose a cumulative exposure hazard. We evaluated the effects of two ATR by-products, diamino-s-chlorotriazine (DACT) and hydroxyatrazine (OH-ATR), and a structurally similar chlorotriazine, propazine (PRO), on female pubertal development. Rats were gavaged from postnatal days (PNDs) 22 through PND 41 with DACT (16.7, 33.8, 67.5, 135 mg/kg), OH-ATR (22.8, 45.7, 91.5, 183 mg/kg), or PRO (13, 26.7, 53, 106.7, 213 mg/kg). The dose range for each chemical was selected as the molar equivalent of ATR (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal opening (VO) and killed on PND 41. DACT, a by-product of ATR that occurs in the environment and is also the primary chlorinated metabolite of ATR in animal tissue, delayed VO by 3.2, 4.8, and 7.6 days compared to the controls (33.1 +/- 0.4 (SE) days of age) following exposure to 33.8, 67.5, and 135 mg/kg, respectively. The no effect level (NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for ATR (25 mg/kg). Although the body weight (BW) on PND 41 was reduced by the high dose of DACT (8.4% reduction), this reduction did not exceed the criteria for selecting the maximum tolerated dose (e.g., a dose that causes >10% decrease in BW at necropsy). None of the lower doses of DACT caused a significant difference in BW gain. Additionally, 33.8, 67.5, and 135 mg/kg of DACT significantly increased the BW on the day of VO. PRO (107 or 213 mg/kg) delayed VO by 4 days but did not alter the BW on PND 41. While no significant delays in pubertal development were observed in two separate dose-response studies with doses ranging up to 183 mg/kg (OH-ATR), a minor but statistically significant delay in the onset of puberty in a pilot study using OH-ATR raises the possibility that an effect might occur following exposure to higher doses. However, it is clear from these data that OH-ATR has a much lower potency when compared with equimolar doses of DACT and PRO. Together, these data demonstrate that PRO and DACT can delay the onset of puberty in the female rat at doses equimolar to ATR and provide the scientific basis for the use of additivity in the upcoming risk assessments.