Both CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta are intronless, yet can create various N-terminally truncated protein products with distinct DNA binding and transactivation potentials. These proteins can be generated via two distinct mechanisms, one translational and the other post-translational. In the translational mechanism, there is alternative translational start site selection of the different AUG codons present in the single messenger RNA (mRNA) species via a process of leaky ribosome scanning. Additionally, a post-translational method of isoform formation, through specific proteolytic cleavage of the full length protein has also been described. In this manuscript, we present evidence that the production of C/EBPbeta protein isoforms in the neonatal mouse liver is regulated by C/EBPalpha. In C/EBPalpha knockout mice, the predominant C/EBPbeta proteins are the larger 38- and 35-kd isoforms, whereas wild-type animals primarily possess the smaller 21- and 14-kd isoforms. These C/EBPalpha-dependent differences are liver specific, not present in lung or adipose tissues, and present at day 18 of development. Additionally, we show that induction of C/EBPalpha expression leads to an increase in the production of the 21-kd C/EBPbeta isoform in cell culture studies. As the various C/EBPbeta protein isoforms have different transcriptional capabilities, it is important to understand the regulation of the production of these isoforms. Our observations suggest a novel role for the C/EBPalpha transcription factor in this process.