CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcriptional regulatory factor that inhibits cell proliferation, and alternative translational initiation produces two polypeptides, C/EBPalphap30 and C/EBPalphap42. By expression profiling, it was revealed that C/EBPalphap30 specifically inhibited a unique set of genes, including MPP11, p84N5 and SMYD2, which were not affected by C/EBPalphap42 in both QSG-7701 hepatocyte cell line and QGY-7703 hepatoma cells. Semi-quantitative RT-PCR analysis independently confirmed these results. Chromatin immunoprecipitation assay showed that C/EBPalphap30 bound to the promoters of these genes more strongly than C/EBPalphap42. In clinical hepatoma samples in which C/EBPalpha was downregulated, all three genes specifically inhibited by C/EBPalphap30 were upregulated. However, repression of MPP11, p84N5 and SMYD2 genes might not be directly involved in C/EBPalphap30-mediated growth inhibition. Our data suggest that C/EBPalphap30 regulates a unique set of target genes and is more than a dominant-negative regulator of C/EBPalphap42.