CCAAT/enhancer-binding protein (C/EBP) alpha is a bZIP transcription factor whose expression is restricted to specific cell types. Analysis of C/EBPalpha mRNA and protein levels in various mammalian cells indicates that expression of this gene is controlled both transcriptionally and post-transcriptionally. We report here that C/EBPalpha translation is repressed in several cell lines by an evolutionarily conserved upstream open reading frame (uORF), which acts in cis to inhibit C/EBPalpha translation. Mutations that disrupt the uORF completely abolished translational repression of C/EBPalpha. The related c/ebpbeta gene also contains an uORF that suppresses translation. The length of the spacer sequence between the uORF terminator and the ORF initiator codon (7 bases in all c/ebpalpha genes and 4 bases in c/ebpbeta homologs) is precisely conserved. The effects of insertions, deletions, and base substitutions in the C/EBPalpha spacer showed that both the length and nucleotide sequence of the spacer are important for efficient translational repression. Our data indicate that the uORFs regulate translation of full-length C/EBPalpha and C/EBPbeta and do not play a role in generating truncated forms of these proteins, as has been suggested by start site multiplicity models.