Abstract In order to evaluate the substance-specificity of ultrastructural criteria in isolated fish hepatocytes as endpoints in toxicological studies, liver cells isolated from rainbow trout ( Oncorhynchus mykiss) by collagenase perfusion were exposed in vitro to sublethal concentrations of dinitro-o-cresol (DNOC: 0, 0.7, 7.2, 72.2 μmol/1) and 2,4-dichlorophenol (2,4-DCP: 0, 6.1, 61.3, 613 nmol/1) for 1–5 days. With either toxicant, earliest cytological modifications could be detected after 1 day at the lowest test concentrations. Ultrastructural analysis documents a complex, substance-specific combination of dose- and time-dependent effects. Evaluation of single cytological alterations induced by DNOC and 2,4-DCP reveals that each cytopathological reaction consists of both unspecific and substance-specific changes. Unspecific symptoms comprise disturbance of intracellular compartmentation, condensation, marginalization and redistribution of heterochromatin, reduction and reorganization of RER cisternae, proliferation of lysosomes, glycogenosomes, myelinated bodies and large electron-lucent vacuoles with heterogeneous contents, as well as glycogen depletion. Transformation of Golgi cisternae into concentric membrane whorls is specific to intoxication by DNOC, whereas the reaction to 2,4-DCP is characterized by unusual heterochromatin distribution patterns and formation of mitochondrion aggregations. Due to their general character, unspecific changes are recommended as rapid biomarkers of contamination by chemicals, whereas specific changes might be useful for the differential diagnosis of the toxicants.