The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named p45, of the 265 proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 406 amino acid residues with a calculated molecular weight of 45770 and isoelectric point of 8.35. The sequences of several fragments of bovine p45, determined by protein chemical analyses, spanning 27% of the complete structure, were found to be in excellent accord with those deduced from the human cDNA sequence. Computer analysis showed that p45 belongs to a family of putative ATPases which includes regulatory components of 26S proteasomes. The overall structure of p45 was found to be homologous to that of yeast Suglp, which has been identified as a transcriptional factor. It is closely similar, but not identical to the sequence reported for Tripl, a functional homolog of Suglp in human tissues. These results are consistent with the possibility that Sugl-like proteins with distinct sequence function in transcription and protein degradation in human cells. However, the alternative hypothesis, that the same gene locus encodes both p45 and Tripl, cannot be excluded on the basis of such closely similar sequences. In either case, both proteins are likely to function equivalently well in either transcription or protein degradation.