A large data set on Ontario lakes and their fish species was examined for evidence of the influence on species richness of regional, local, anthropogenic, and methodological factors. Analysis of regional species distributions and associations showed patterns consistent with species invasion into Ontario since the last period of glaciation. A comparison of local species richness (mean richness among lakes) with regional richness pointed to a dominance of local over regional factors in determining lake species richness. A multiple regression model of species richness accounted for 48% of variance. Of the two regional factors included in the model, watershed species richness increased lake species richness and elevation decreased it. An increase in two local abiotic factors, lake area and mean depth, increased richness. Richness also increased with increasing pH, an anthropogenic factor, and increased in more recent survey years, an indicator of methodological effects. Analyses of mean species richness in relation to the presence or absence of individual species provided further evidence of sampling (methodological) effects. Overall, the results were consistent with the existing theory of island biogeography and its specific application to fishes in lakes.