A strain of Alcaligenes faecalis T1, which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-100 filtration, and 50 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigera 1-16-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. Km values were 13.3 micrograms/ml (= 0.78 microM, based on an estimated average molecular weight of 17 000) for poly(3-hydroxybutyrate) and 5.4 mM for the trimeric ester of D(--)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of D(--)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing D(--)-3-hydroxybutyrate dimer units one at a time.