The cellular oncogene c-myc encodes a nuclear protein that is considered to play a role in cell proliferation. In this report, the region upstream from the transcriptional promoter of the c-myc gene was examined for regulatory activity on its expression during cell cycle. Plasmids which contain the upstream region of human c-myc gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected to rat 3Y1 cells together with pSV2Hg (containing the hygromycin resistance gene linked to the SV40 promoter). Stably transformed cell lines were obtained by hygromycin selection. In random culture, the cells possessing CAT gene preceeded by the upstream region of the c-myc gene, including the HindIII-PstI [myc(H-P)] region, showed strong CAT activity. The myc(H-P) region contains a c-myc protein complex binding site. On the other hand, the cells carrying a similar myc-CAT construct, but without the myc(H-P) region, showed very low levels of CAT expression. These cell lines were then synchronized by serum starvation and their CAT expression was examined by Northern blotting. The expression became maximal between G1 and S phases of the cell cycle, in correspondence with the increase of endogenous c-myc expression. CAT expression of the cells containing the CAT gene linked to the SV40 enhancer/ promoter was less affected by cell cycle, neither was the expression of a housekeeping gene, the hypoxanthine phosphoribosyl transferase (HPRT). These results suggest that the myc(H-P) region is important for cell cycle dependent regulation of c-myc expression.