The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 18.104.22.168) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.