Mouse F9 teratocarcinoma cells converted into primitive endoderm and parietal endoderm-like cells when treated with retinoic acid (RA) and RA plus dibutyryl cyclic AMP (dbtcAMP), respectively. The carbohydrate chains of glycoconjugates are known to undergo rapid changes during F9 cell differentiation. The mechanism of gene regulation of β1,4-galactosyltransferase (β1,4GalT), one of the glycosyltransferases involved in the synthesis of carbohydrate structures, was explored during the differentiation of F9 cells. Northern blot analysis revealed that the amount of β1,4GalT mRNA increased ∼1.5- and 6.5-fold in response to treatment with RA alone and RA plus dbtcAMP (RA/dbtcAMP), respectively, for 8 days. β1,4GalT specific activity also gradually increased up to 21-fold in response to treatment with RA/dbtcAMP for 8 days. The reason for the different rates of increase in mRNA and enzyme activity remains to be determined. The transcriptional activity of the β1,4GalT gene was measured during the course of RA/dbtcAMP-induced F9 cell differentiation in transient transfection experiments using 5′-upstream region DNA (1.8 kb) of the mouse β1,4GalT gene combined with luciferase cDNA. Although activity was slightly enhanced on the first day after induction, no significant rise in transcriptional activity was observed in the late stage of induction (3–6 days), when mRNA levels were greatly increased. This was further supported by the nuclear run-off assay which indicated that the rate of de novo synthesis of the β1,4GalT gene transcript in the RA/dbtcAMP-induced cells was almost the same as in undifferentiated F9 cells. Measurement of the stability of β1,4GalT transcripts revealed that their half-life gradually increased as the duration of RA/dbtcAMP exposure increased. Two β1,4GalT mRNA isoforms detected by RNase protection assay, a long form (4.1 kb) and a short form (3.9 kb), were found to stabilize at the same rate during RA/dbtc-induced F9 cell differentiation, indicating non-selective regulation of the two isoforms. Considered as a whole, the results of the present study demonstrate that β1,4GalT mRNA accumulates in F9 cells during the course of RA/dbtcAMP-induced differentiation as a result of post-transcriptional stabilization, and not due to transcriptional activation.