The biosynthesis, proteolytic processing, and transport of lysosomal acid phosphatase in normal and I-cell human skin fibroblasts was studied by metabolic labeling of the cells and isolation of acid phosphatase by immunoprecipitation. Several forms of the enzyme were identified in pulse-chase experiments. The largest precursor form had a Mr of 110,000. It was accompanied by several smaller polypeptides (Mr = 84,000-62,000), which were localized to light membranes containing the markers of endoplasmic reticulum and Golgi complex. These polypeptides were further processed to mature forms with apparent Mr of 57,000, 48,000, and 43,000 that accumulated in the cells and were associated with dense lysosomes. Less than 10% of newly synthesized acid phosphatase was secreted mainly as Mr = 112,000 and 74,000 forms. The processing of acid phosphatase was inhibited by NH4Cl and by a peptidyldiazomethyl ketone inhibitor of cysteine proteinases. The intracellular Mr = 110,000, 57,000, and 48,000 and the secreted Mr = 112,000 and 64,000 forms contained phosphorylated oligosaccharides cleavable by endo-beta-N-acetylglucosaminidase H. Transport of acid phosphatase into lysosomes was sensitive to NH4Cl and dependent on mannose 6-phosphate specific receptors by the following criteria: (i) inhibition of endocytosis of acid phosphatase by mannose 6-phosphate, (ii) enhancement of the secretion of acid phosphatase in the presence of antibodies to the mannose 6-phosphatase specific receptor, and (iii) secretion of about two-thirds of newly synthesized acid phosphatase in I-cell fibroblasts. Obviously, the mechanism of transport of acid phosphatase into lysosomes is indistinguishable from that operating for other lysosomal enzymes in fibroblasts. In contrast to other lysosomal enzymes, acid phosphatase appears to be subjected to an early proteolytic processing, presumably within the endoplasmic reticulum, which results in secretion of several processed forms of the enzyme.