Inbred strains of mice exhibited significant variation in whole blood galactokinase (GALK) activity. Activities tend to cluster into two classes, one class having approximately twice the activity of the other. Hybrids (F1) between high and low strains have activity intermediate between the parental activities. Two sets of recombinant inbred (RI) lines were developed by brother-sister mating, beginning with F2 generations of crosses between different pairs of high and low GALK activity strains. The RI lines segregated in terms of GALK activity, indicating single gene inheritance; this galactokinase locus has been designated Glk. The strain distribution patterns of both RI series agreed closely with esterase-3 (Es-3) alleles of the respective parental strains (31/34 independently derived strains were concordant for Es-3 and Glk genotypes), a finding consistent with a map distance between loci of 2.5 cM. Es-3 has been located on the distal end of chromosome 11. Glk, with its alleles Glka (lower activity) and Glkb (higher activity), is therefore assigned to the same region.