Effluent from eight different pulp and paper mills was screened for endocrine disrupting activity using a series of human and fish based in vitro assays. Effluent grabs (1 L) were extracted sequentially with dichloromethane and methanol. Estrogen and Ah receptor gene expression assays, vitellogenin induction, estrogen (ER) and androgen receptor (AR) and sex steroid binding protein binding (SSBP) affinity were used. All ER-mediated screening assays concurred that treated and untreated effluent from a thermomechanical (TMP) mill possessed dose-dependent estrogenic activity. There was some discrepancy between estrogenic screening assays, where vitellogenin induction and hepatic trout ER binding identified activity in seven mills. The estrogen equivalence in effluents for all assays ranged from 1.3-738 ng E2/L effluent. Although the [³H]-testosterone competitive AR binding assay revealed weak ligands in the TMP effluent, two additional mills possessed androgenic activity. These results were in agreement with the human and goldfish SSBP assays, which identified [³H]-E2 and [³H]-dihydroytestosterone ligand competitors in the same three effluents. Generally, treated versus untreated effluent, tended to have less endocrine disrupting activities, where the level of estrogen equivalence or AR binding affinity was decreased to lower levels. In summary, in vitro tests revealed that effluents from 8 different pulp and paper mills (TMP and bleached kraft mill) had constituents with significant endocrine disrupting activity.