In the emerging field of mechanistic toxicology, growing attention is being paid to the interpretation of sex-related toxic responses. The Wistar rat outbred stock is a frequently used rodent for toxicity testing. Outbred strains, which display a relatively high degree of genetic variability between the individual animals are often used in risk assessment, as they are considered to best approximate the variability present in a human population. In this study two-dimensional (2-D) protein gel electrophoresis was applied to investigate the liver protein patterns of male and female Wistar rats and to search for (i) sex-related and (ii) interindividual qualitative and quantitative differences in protein expression. Among the sexes, six proteins were detected that were shown to be exclusively present in male rats and one that was present only in females. A male-specific protein was tentatively assigned to alpha 2u globulin. Seven protein spots showed statistically significant abundance changes (p < 0.001) between males and females, one was tentatively assigned to heme oxygenase 1 and another to the 23 kDa morphin-binding protein. Four sets of protein spots were detected that showed positional shifts in the individual patterns and are likely to represent polymorphic proteins inherent in the Wistar rat. These results form a valuable basis for future investigations of drug-induced changes in the male and female Wistar rat liver pattern.