Cyclin D1, DNA topoisomerase I, and proliferating cell nuclear antigen (PCNA) are three important cell cycle regulatory proteins. Recently, their promoters have been isolated, thus facilitating molecular analysis of transcriptional control mechanisms of these genes. Transcription of these three promoters in stable K562 transfectants during different cell cycle phases was analyzed after cell cycle synchronization. About 1 kb of 5' flanking region from either cyclin D1 or DNA topoisomerase I gene is sufficient to confer G1- or S-phase-specific transcription activity to chloramphenicol acetyltransferase (CAT) reporter genes, respectively. In contrast, 2.8 kb of 5' flanking sequences from the PCNA gene led to constitutive transcription, but the inclusion of a segment of the PCNA gene first intron, which contains evolutionarily conserved sequences, could enhance transcription in G1/S-enriched nuclei. This PCNA intron region contains a binding site recognized by the transcription factor E2F. To test whether this site is functional, we cotransfected PCNA-CAT genes with E2F-1 and DP-1 expression plasmids. Expression of the E2F-1/DP-1 heterodimer activated the CAT gene with the PCNA intron. Therefore, this intron region, involved in transcriptional activation at the cell cycle G1/S boundary, is also E2F inducible.