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Past studies at a bleached kraft pulp mill located in Saint John, NB, Canada have shown that chemical recovery condensates significantly depress circulating and gonadal steroids in mummichog (Fundulus heteroclitus), an endemic fish species. In the present study, compounds were extracted from the condensates, and a toxicity identification evaluation (TIE) was conducted to characterize the hormonally active substances (HASs) in the condensates. Extracts were fractionated by high performance liquid chromatography (HPLC) and mummichog were exposed to the fractions in a 7-day bioassay. Plasma testosterone was measured for each sex following exposure. Responses in fish exposed to the whole extract at 1% v/v were not as pronounced as had been observed previously (female plasma testosterone was reduced by 16% in the current study compared to 75% previously). Dose–response experiments showed an exposure concentration of 4% v/v was required to elicit significant plasma steroid reductions. Despite these responses, individual condensate fractions actually increased steroid levels in mummichog, which suggests that multiple HASs may need to act synergistically or additively to elicit effects, and if separated, the compounds may have different hormonal activity. The HASs in question caused a reduction in male gonad size at 4% v/v, and have sex-dependent mechanisms of action (males were more responsive to exposure than females). [Copyright &y& Elsevier]