Bile canalicular phosphatase activity in frog, chicken, rat and cat has been studied with respect to substrate specificity, pH optimum and effect of various stimulators and inhibitors. It is concluded that three different bile canalicular phosphatase activities may be demonstrated histochemically: 1. A strong non-specific nucleoside diphosphatase able to split ATP and most diphosphates. Its relevance is, however, uncertain and requires confirmation by biochemical studies. 2. A non-specific alkaline phosphatase, 3. 5′-nucleotidase. Evidence is presented that the bile oanalicular staining reflects real enzyme activity and not artificial non-enzymatic hydrolysis.