We have developed a new procedure for the selective determination of β1‐3 and β1‐4 galactosyltrans‐ferases with Lc 3Cer as the substrate and the microsomes of fetal and adult porcine livers as the enzyme sources. This method was based on the detection of such products as Lc 4Cer for β1‐3 galactosyltransferase (β1‐3GT) and nLc 4Cer for β1‐4 galactosyltransferase (β1‐4GT), with monoclonal anti‐Lc 4Cer and anti‐nLc 4Cer antibodies, respectively. This method thus enabled us to differentiate the activity of β1‐3GT from that of 4bT1‐4GT with a high degree of sensitivity. The method was then used to determine the activities of both enzymes in human gynecological carcinoma‐derived cells. Four of the five cell lines derived from uterine endometrial cancer expressed significantly high levels of specific activity of β1‐3GT among the cell lines examined, while their β1‐4GT activities were less than 20% of that for β1‐3GT in the endometrial carcinoma‐derived cells. On the other hand, a higher specific activity of β1‐4GT than that of β1‐3GT was detected in the cell lines derived from uterine cervical and ovarian cancers. These findings were thus found to correlate closely with the rate of expression of Lc 4Cer‐ and nLc 4Cer‐based carbohydrate chains in the cell lines based on the results of immunohistochemical staining.