A method is presented for detection of lysosomal hydrolase activity in situ in colonies of Chinese hamster ovary cells. This method was used to screen for mutants deficient in lysosomal alpha-mannosidase. Mutagenized cells were replicated onto filter papers [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] and allowed to divide for 11-14 days; cells on the filter replicas were lysed, and the filters were incubated with 4-methylumbelliferyl-alpha-D-mannopyranoside at pH 4.4 and then photographed under UV light. Colonies for which the replicas exhibited little or no fluorescence were picked from the master plates and purified; mutants with decreased alpha-mannosidase activity were obtained at a frequency of 1 per 1500 mutagenized cells. Analysis of one of these mutants showed that lysosomal alpha-mannosidase activity was 18% of that from wild-type cells, whereas the activities of alpha-mannosidases not of lysosomal origin were similar in the wild type and mutant (these included both Golgi-associated and cytosolic enzymes as well as a novel acid alpha-mannosidase seen only in cells from confluent culture). The mutant contained normal levels of other lysosomal hydrolases. Both wild-type and mutant cells secreted alpha-mannosidase into the medium at levels proportional to those found inside the cells. Incubation of mutant cells with secretions from the wild type resulted in partial restoration of enzyme activity. Added enzyme was localized in the lysosomal fractions; uptake of added enzyme was inhibited by mannose 6-phosphate and fructose 1-phosphate, which are known to inhibit uptake of lysosomal enzymes into human diploid fibroblasts.