The effects of 6-ethoxyzolamide (ETZ), a carbonic anhydrase (CA) inhibitor, on differentiation of the mouse preadipocyte cell line 3T3-L1 were examined by quantitative image analysis of intracellular fat storage. For adipocyte differentiation, postconfluent cells were treated with a ligand of the peroxisome proliferator-activated receptor gamma (PPARgamma), troglitazone (TRG), and dexamethasone for 2 days. Differentiated cells showed weak fat staining at day 4 which increased thereafter, correlating with CAIII expression. ETZ treatment for 10 days at a 200microM concentration reduced both the percentage of differentiated adipocytes storing fat and the amount of fat stored in individual cells. These findings were also supported by the results of fluorescence activated cell sorting analysis. Despite their light fat staining, however, CAIII was not expressed in ETZ-treated cells. Furthermore, ETZ delayed the clonal expansion of cells, an early event preceding differentiation. Northern blot analysis revealed a high CCAAT/enhancer binding protein (C/EBP) beta mRNA level and low PPARgamma mRNA in ETZ-treated cells. Thus, increased C/EBPbeta mRNA did not lead to enhanced PPARgamma expression in this case. Another CA inhibitor acetazolamide did not inhibit adipocyte differentiation, although the drug exhibited a similar inhibition pattern for CA activity as ETZ. These results suggested that inhibitory effects of ETZ on adipocyte differentiation were not due to inhibition of CA activity but rather due to altered levels of the transcription factors.