The intracellular distribution and level of acid hydrolases in Ochromonas malhamensis were studied in cells grown osmotrophically in a defined medium, in a carbon-free starvation medium, and during phagotrophy in each of these media. By cytochemical techniques, little enzymic reaction product was observed in the vacuoles of osmotrophic cells grown in the defined medium. Starved cells, however, contained autophagic vacuoles and cannibalized other Ochromonas cells. Dense enzymic reaction product was observed in the digestive vacuoles and in the Golgi cisternae of these starved cells. Moreover, starved cells and cells grown in a nutritionally complete medium ingested Escherichia coli which appeared in digestive vacuoles containing enzymic reaction product. Biochemical assays for lysosomal acid phosphatase (E.C. 188.8.131.52 orthophosphoric monoester phosphohydrolase) and acid ribonuclease (E.C. 184.108.40.206 ribonucleate nucleotido-2'-transferase) were done on Ochromonas cultures in the same experimental treatments and under identical assay conditions as the cytochemical study. During starvation, the acid hydrolase specific activities were consistently twice those found in cells grown in an osmotrophic complete medium. Ochromonas fed E. coli showed no increase in acid hydrolase specific activity as compared to controls not fed E. coli. The latency of lysosomal acid hydrolases in cells fixed with glutaraldehyde was reduced, suggesting that this fixative increases lysosomal membrane permeability and may release enzymes or their reaction products into the cytoplasmic matrix during cytochemical analysis. This could explain the cytoplasmic staining artifact sometimes observed with glutaraldehyde-fixed cells when studied by the Gomori technique. This study confirms that Ochromonas malhamensis, a phytoflagellate, does produce digestive vacuoles and can ingest bacteria, thereby fulfilling its role as a heterotroph in an aquatic food chain. When Ochromonas is grown in a nutritionally complete osmotrophic medium, phagocytosis causes appearance of acid hydrolases in the digestive vacuoles, whereas the total activity of the enzymes remains unchanged. An organic carbon-free medium strongly stimulates acid hydrolaes activity and causes these enzymes to appear in the digestive vacuoles whether phagocytosis occurs or not.