The mechanisms of action of an estrogenic chemical have been examined in a viviparous fish the eelpout (Zoarces viviparus), by identification of an upregulated estrogenic pathway--the induction of hepatic estrogen receptor mRNA, hepatic estrogen binding activity and plasma vitellogenin. A relative quantitative RT-PCR assay has been established to measure hepatic estrogen receptor alpha (ER) mRNA levels in eelpout. Assay conditions were optimised using control and induced samples to ascertain its applicability in the actual working range of ER mRNA concentrations. beta-Actin was co-amplified and used as an internal standard. Time-course effects of water exposure to 0.5 microg/L 17beta-estradiol (E(2)) and 25 microg/L of the xeno-estrogen 4-tert-octylphenol (4-tert-OP) on ER mRNA levels in the male eelpout was examined. After 48 h of exposure, ER transcripts were induced 15-fold and 6-fold in the E(2)- and OP-treated fish, respectively. This difference, however, was not apparent after 1 week of exposure, when similar high levels of ER mRNA were present in both groups (20-fold induction). This indicates that the estrogenic capacity of 4-tert-OP increases with exposure time. The effect of treatment was also evaluated by examining the induction of specific E(2) binding capacity in hepatic cytosolic extracts and by measuring vitellogenin in plasma. Both parameters were also induced by the treatments, but later in the time course. The measurement of ER mRNA by the RT-PCR assay showed to be the most sensitive method for the detection of estrogenic responses in eelpout.