To understand fifth complement component (C5) gene regulation, splicing, and C5 protein deficiency at the molecular level, the organization of the murine C5 gene was determined. The C5 structural gene is present as a single copy in the mouse genome as demonstrated by Southern blot analysis. Accordingly, three cosmid clones were isolated from a genomic library that was prepared from mouse strain B10.D2/nSnJ. These clones overlapped and contained the structural gene encoding the complete C5 alpha-chain and 90% of the beta-chain. The 5'-flanking region of the C5 gene was obtained from a clone isolated from a genomic lambda-MOPC-41 library. Unique restriction fragments were prepared from the genomic clones and subcloned, and the exons were sequenced. All introns were sized by sequencing or Southern analysis. The C5 structural gene was found to be a highly interrupted gene of approximately 78 kilobases containing 42 exons and 41 introns. The exons ranged in length from 58 to 247 base pairs, with an average length of 131 base pairs. The introns ranged in size from 100 base pairs to 4 kilobases with an average length of 1.5 kilobases. The C5 alpha-chain was encoded by 49 kilobases containing 26 exons; the beta-chain was encoded by 29 kilobases containing 16 exons. The C5a coding sequence was split between two exons. All intron/exon junctions followed the normal consensus rule except at intron 35 in which the 5'-donor GT was substituted by GC. The 2-base-pair gene deletion and HindIII and PvuII restriction fragment length polymorphisms associated with murine C5 deficiency were localized to exon 7, exon 16, and intron 20, respectively. Comparison of the intron-exon junctions of the murine C5, human C3, and mouse C4 genes indicated that these genes are nearly identical in structural organization. However, the rat alpha 2-macroglobulin gene showed only moderate genomic organizational similarity to the murine C5 gene. A major and a minor transcriptional initiation site in the C5 gene were identified by primer extensions and confirmed by RNase protection assays. Sequence analysis of the 5'-flanking region (760 base pairs) revealed a TATA-like and CAAT box upstream of the major transcriptional initiation site at positions -274 and -303, respectively, suggesting an atypical promoter. The 5'-flanking region also contained sequences identical with several cis-acting motifs known to bind the liver-specific nuclear protein LF-A1 and the nuclear protein NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)