The androgenic potential of a New Zealand pulp mill effluent (PME) and a Canadian PME was assessed along with a New Zealand sewage treatment plant effluent (STP) using a combination of in vivo and in vitro methods. The in vitro methods included: (1) a fish-based androgen receptor binding assay, (2) a fish-based aromatase inhibition activity assay, and (3) an analysis of gonadal sex steroid levels in exposed female mosquitofish ( Gambusia affinis ) ovaries by radioimmunoassay. The in vivo method included a quantifiable analysis of anal fin ray length for female mosquitofish exposed to the effluents. Effluent extracts for the Canadian PME and New Zealand STP were found to have low in vitro androgenic potential compared to upstream reference extracts. All effluent extracts (Canadian PME, New Zealand PME and STP) showed a low degree of in vitro aromatase inhibition potential compared to upstream reference extracts. In vivo analysis showed no androgenic potential of the New Zealand PME and STP. The in vitro androgen receptor assay and in vivo mosquitofish bioassay did show androgenic responses for androstenedione (AD) and 1,4-androsta-diene-3,17-dione (ADD), which are two products of the microbial conversion of β-sitosterol (a plant sterol commonly found in PME) by Mycobacterium smegmatis . Also, the potential of the mosquitofish bioassay to determine anti-androgenic effects in effluents was demonstrated.