UDP-D-Galactose:D-xylose galactosyltransferase (Galactosyltransferase-I) is an enzyme involved in the synthesis of the linkage region of chondroitin sulfate. Measurement of galactosyltransferase-I in mature articular cartilage has depended on milling such tissue in liquid nitrogen to break open the cells and enable interaction of exogenous substrates with galactosyltransferase-I. This process requires large amounts (approximately 1 g wet wt) of cartilage. This paper reports the development of an assay for galactosyltransferase-I in articular cartilage where 10-25 mg (wet wt) of the tissue was extracted with a buffer containing the detergent Thesit. This assay was more time efficient, required less tissue, and was linear for up to 6 h. It was effective on both mature bovine articular cartilage and embryonic chick epiphyseal cartilage, detecting 100 and 68% of total galactosyltransferase-I activity, respectively. The higher number of measurements available allowed kinetic studies to be conducted on bovine articular cartilage explant cultures under conditions of up- and down-regulation. Incubation with fetal calf serum resulted in an increase in galactosyltransferase-I activity. Galactosyltransferase-I activity, however, did not change markedly in cartilage cultured in the absence of fetal calf serum or in the presence of cycloheximide. A soluble preparation of galactosyltransferase-I, extracted from bovine articular cartilage using Thesit, eluted from a Superose-6 column, with a molecular mass of about 55 kDa.