A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purified to homogeneity from Rhodotorula glutinis 38B1, whose resting cells have been previously reported as useful for the conversion of 7-ACA derivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667-2672, 1996]. The purified enzyme was a dimer comprised of identical subunits with a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low K-m and high k(cat) values as substrates, as well as some acetyl esters with relatively long-chain alcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most active at 35 degrees C and pH 5.5, and was inhibited by several serine enzyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked "hybrid type" oligosaccharide, and most of the enzyme activity was found in the purified cell wall fraction. The enzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.