Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the -galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased -galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 -galactosidases. Similar functions for other -galactosidases in both GHFs 2 and 42 are suggested by genomic data.-Galactosidases, such as the LacZ -galactosidase of Escherichia coli, are used as molecular biology tools, industrial enzymes, and models for exploring structure-function relationships. However, these uses do not often yield insight into the physiological roles of these enzymes. Although -galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 -galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33, 47, 65), hypersaline environments (27, 54), and soil (Bacillus and Streptomyces spp.).Over a dozen GHF 42 enzymes have been characterized, and most are -galactosidases (as shown by maximal activity with the) with less than 10% relative activity on nongalactosidic chromogens (with two exceptions [27,34]). However, experimental data supporting lactose hydrolysis by GHF 42 -galactosidases are absent (34, 44, 48) or weak. Several prokaryotes possessing a GHF 42 gene are unable to grow on lactose as a sole carbon source (1, 9, 27), and at least two GHF 42 -galactosidases do not cleave lactose in vitro (27,64). The determination of growth on lactose can be complicated by the presence of multiple -galactosidases (7,16,25) because not all of the -galactosidases may be participating equally (or at all) as...