Glutamine synthetase (GS) activity increases more than 100-fold during adipocyte differentiation of cultured 3T3-L1 cells. We now find that Northern hybridization analysis of RNA from 3T3-L1 adipocytes with a rat GS cDNA clone (pGSRK-1) yields two hybridizable GS RNAs of length 3.2 and 1.6 kilobases (kb). Densitometric analyses of autoradiographs of the Northern blots probed with pGSRK-1 indicate that the 3.2 kb GS-specific RNA is at least 4- to 5-fold more abundant than the 1.6 kb GS RNA. Analyses of both total and poly(A+)RNA from 3T3-L1 adipocytes yielded similar results. (It is noteworthy that an mRNA of 1.2 kb would be sufficient to encode the 42 500 Da GS subunit.) Quantitative dot-blot hybridization analysis indicates that dexamethasone increases GS mRNA while both insulin and dibutyryl cAMP decrease GS mRNA and/or prevent the dexamethasone-mediated increase. Our data suggest that there are at least two GS mRNAs in 3T3-L1 adipocytes and that they are regulated in parallel by dexamethasone, insulin and dibutyryl cAMP.