In search for nuclear proteins that interact with the human thymidine kinase (htk) promoter, we discovered that p37AUF, a hnRNP C‐like protein, and hnRNP A1, both members of the heterogeneous ribonucleoprotein family, can bind with high affinity to an ATTT sequence motif contained within the cell cycle regulatory unit (CCRU). We report here that over‐expression of p37AUF stimulates gene expression mediated by the htk promoter in a promoter‐sequence specific manner, whereas hnRNP A1 suppresses it. Both recombinant p37AUF and hnRNP A1 can bind the htk CCRU, suggesting that their binding to the DNA target does not require additional cellular components. We further discovered that hnRNP K is a potent suppressor of htk mediated gene activity. However, its mechanism of action is mediated through protein‐protein interaction, since hnRNP K itself cannot bind the htk CCRU but can competitively inhibit the binding of other hnRNPs. The binding site for the hnRNPs on the htk CCRU is not required for S‐phase induction of the htk promoter. However, in stable but not transient transfectants, the mutation of the hnRNP binding site results in 5‐ to 10‐fold reduction of htk mediated gene activity in synchronized and exponentially growing cells. Collectively, these findings support emerging evidence that hnRNPs, in addition to their traditional role in RNA biogenesis, could be regulators of gene expression through direct DNA binding or interaction with other proteins. J. Cell. Biochem. 79:395–406, 2000. © 2000 Wiley‐Liss, Inc.