The aim of this study was to address whether there is a fundamental difference in regulation of histone gene expression in cells that have become quiescent but retain the ability to proliferate, compared with those cells that have differentiated. We compared multiple levels of regulation of histone gene expression during 3T3-L1 pre-adipocyte differentiation. Confluent cells induced to differentiate by treatment with insulin, dexamethasone, and isobutylmethylxanthine initially exhibited an increased proliferative response compared with cells given serum alone. This initial differentiation response was associated with a twofold increase in both histone gene transcription and cellular histone mRNA levels, as well as with enhanced sequence-specific binding of nuclear factors to the proximal cell-cycle-regulatory element of the H4 histone promoter. Transforming growth factor beta 1, an inhibitor of 3T3-L1 differentiation, increased both the percentage of proliferating cells and the cellular levels of histone mRNA when given in addition to serum stimulation, but no enhancement of these parameters was observed upon addition of TGF beta 1 to the differentiation treatment. Interestingly, although TGF beta 1 enhanced binding of nuclear factors to the proximal cell cycle regulatory element of the histone promoter, these protein/DNA interactions were not associated with an increase in histone transcription. Our results are consistent with the down-regulation of histone gene expression at confluency being controlled primarily at the post-transcriptional level, in contrast to an increased involvement of transcriptional down-regulation at the onset of differentiation.