Six overlapping clones, spanning the entire C3 gene and the 5' flanking region, were isolated from two genomic lambda libraries. Thirty exons, covering the complete beta chain and part of the alpha chain as far as the C3d region, were analyzed. The full exon-intron organization of the gene was deduced by combining our data with the reported organization of the alpha' chain (Barnum et al., 1989, J. Biol. Chem. 264: 8471-8474). The complete gene is 41 kb and consists of 41 exons. The C3 beta chain spans 13 kb from exon 1 to exon 16. Exon 16 encodes both alpha and beta chains. The alpha chain is 28 kb and contains 26 exons, including exon 16. The 5' flanking region was sequenced up to 514 bases upstream from the ATG start site. The major transcription initiation site was mapped to an adenine residue 61 bases upstream from the signal peptide by primer extension analysis of poly(A)+ mRNA from hepatocytes and U937 cells. The TATA box motif was assigned at position -85. Several putative binding sites for transcription factors were found in the 5' flanking region, suggesting possible pathways for the regulation of the C3 gene.