Extensive global use of the serotonin norepinephrine reuptake inhibitor Amitriptyline (AMI) for treatment of mental health problems has led to its common occurrence in the aquatic environment. To assess AMI bioconcentration factors, tissue distribution, and metabolite formation in fish, we exposed gilt-head bream (Sparus aurata) to AMI in seawater for 7 days at two concentrations (0.2 mu g/L and 10 mu g/L). Day 7 proportional bioconcentration factors (BCFs) ranged from 6 (10 mu g/L dose, muscle) to 127 (0.2 mu g/L dose, brain) and were consistently larger at the low dose level. The relative tissue distribution of AMI was consistent at both doses, with concentrations decreasing in the order brain approximate to gill > liver > plasma > bile >> muscle. Using a suspect screening workflow based on liquid chromatography-high resolution (Orbitrap) mass spectrometry we identified 33 AMI metabolites (both Phase I and Phase II), occurring mostly in bile, liver and plasma. Ten structures are reported for the first time. Remarkably, all 33 metabolites retained the tricyclic ring structure common to tricyclic antidepressants, which may be toxicologically relevant. Collectively these data indicate that, in addition to AMI, a broad suite of metabolites should be included in biomonitoring campaigns in order to fully characterize exposure in aquatic wildlife.