ABSTRACT.Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions ofParamecium caudalumand ofP. tetraureliawere carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p‐nitrophenylphosphatase (pNPPase), glucose‐6‐phosphatase (G6Pase), and 5′‐nucleotidase (AMPase). The results generally support the idea thatParameciumcells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose‐6‐phosphate (G6P) and adenosine 5′‐monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na+‐K+‐ATPase was observed. Since the hydrolysis of p‐nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay forParameciumAcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction.P. caudatumAcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P<β‐glycerophosphate