We have identified a genetic locus, pho4, in Schizosaccharomyces pombe which encodes a minor expressed cell surface acid phosphatase that is repressed by low concentrations (0.5 microM) of thiamin. The enzyme was purified from a strain that overproduces the enzyme. It is an Asn-linked glycoprotein. Removal of the carbohydrates by endoglycosidase H does not abolish enzymatic activity. The molecular mass of deglycosylated and unglycosylated enzyme that accumulates in membranes when cells are grown in the presence of tunicamycin is 56 kDa as determined by sodium dodecyl sulfate-gel electrophoresis. Thiamin regulation, at least in part, operates by reducing the level of pho4-mRNA. Pho4 is not genetically linked to the phosphate repressible acid phosphatase gene pho1. Phosphate and thiamin repressible acid phosphatase differ in their substrate specificity. Their protein moieties are immunologically related. Pho4 and pho1 are the only genes in S. pombe that express cell surface acid phosphatases being enzymatically active with nitrophenyl phosphate as substrate. S. pombe is not unique in having a thiamin repressible acid phosphatase. In Saccharomyces cerevisiae this enzyme is encoded by PHO3.