Smooth muscle cells were cultured from pig aortas and their acid phosphatase activity toward 4‐methylumbelliferyl phosphate was studied. Cell homogenates hydrolysed this substrate with a pH optimum of 5.0, with very little activity at alkaline pH. The apparent Km of the activity was approximately 20 μM at pH 5.0, and the activity was directly proportional to both the incubation time and to the concentration of homogenate. Acid phosphatase activity was inhibited by p‐chloromercuribenzoic acid, by fluoride ions, and by chloroquine.Postnuclear supernatants of the cells were subjected to analytical subcellular fractionation on sucrose density gradients. Acid phosphatase activity had a modal equilibrium density of 1.13 g/ml, which was similar to that of 5′‐nucleotidase and galactosyltransferase, marker enzymes for the plasma membrane and Golgi complex, respectively. The distribution of acid phosphatase activity also had a shoulder of equilibrium density about 1.16 g/ml, which may have corresponded to activity in the lysosomes. The activity of N‐acetyl‐β‐glucosaminidase, a lysosomal marker enzyme, had a very broad distribution with much of its activity equilibrating at low densities.When the cells were homogenized in sucrose medium containing digitonin, a selective membrane perturbant, the N‐acetyl‐β‐glucosaminidase activity remained almost entirely in the sample layer, showing that the lysosomes had been disrupted. The modal equilibrium density of 5′‐nucleotidase activity was increased to 1.19 g/ml, whereas the modal equilibrium density of galactosyltransferase activity was not significantly affected. Acid phosphatase activity showed a similar increase in modal equilibrium density to 5′‐nucleotidase, but had a shoulder on the least dense side of its peak, which may have corresponded to activity in the Golgi complex. To confirm the plasma membrane localization of a large proportion of the acid phosphatase activity, intact cells were treated with digitonin and were then washed and homogenized in sucrose medium without digitonin. The modal equilibrium densities of both 5′‐nucleotidase and acid phosphatase activity were increased to similar extents, whereas the equilibrium densities of the marker enzymes for the other organelles were not significantly affected.Thus, although acid phosphatase is a widely used marker enzyme for the lysosomes in subcellular fractionation studies, its activity towards 4‐methylumbelliferyl phosphate cannot be used as a marker for these organelles in pig arterial smooth muscle cells in culture.