In phosphorylase assays in crude yeast extracts with glucose-1-phosphate (G-1-P) as substrate, 25-30% of the Pi-liberating activity could not be inhibited by antibodies against yeast phosphorylase and were attributed to the action of phosphatases. During phosphorylase preparation from baker's yeast (Saccharomyces cerevisiae), a phosphatase, molecular weight 45000±5000, with high specificity for G-1-P, pH-optimum 5.6, was isolated which appeared to be responsible for the interference. It did not hydrolyze other glycolytic intermediates, pyrophosphate or adenylates. No activation by Mg2+ or inhibition by (+)-tartrate, and only 40% inhibition by 50 mM F- were observed, 5,5′ dithiobis-(nitrobenzoic acid) (10mM) inactivated the enzyme completely. Its affinity for G-1-P was very low (Km=40 mM). Consequently, it mainly interfered with the phosphorylase assay in the amylose synthesizing reaction, in which high G-1-P-concentrations have to be used. For phosphorylase assays in crude extracts, measurement of the phosphorolytic activity is recommended, in which the concentration of G-1-P is kept sufficiently low. © 1979 Springer-Verlag.