Parturition is preceded by a large increase in gap junctions between myometrial smooth muscle cells. Connexin 43 is the major structural protein of myometrial gap junctions. To explore transcriptional regulation of the myometrial Cx43 gene, we used DNase I footprinting, electrophoretic mobility shift and transient transfection assays to examine a 312 bp promoter region (-164 to +148) of the gene, utilizing human myometrial cell cultures and nuclear extracts. The DNase I studies showed four regions of nucleoprotein interactions. Protection of region 1 (-80 to -31) encompassed an Activator Protein 1 (AP1) (-44 to -36) and two Specificity Protein 1 (Sp1) (-77 to -69 and -59 to -48) consensus sequences. Regions 2 to 4 included the transcription initiation site (-10 to +25), an Ets/NF-kB consensus sequence (+47 to +74) and a TA-rich region (+81 to +101) respectively. Gel mobility shift and supershift assays demonstrated c-Jun and Sp1 binding at the AP1 and Sp1 sites respectively. Promoter mutagenesis and transient transfection analyses combined with Sp1 and c-Jun/c-Fos over-expression studies indicate that both Sp1 and c-Jun are required for maximal promoter activity and, therefore, may positively regulate transcription of myometrial Cx43 during the initiation of labour.