The crucial role of costimulatory molecules, CD28, CTLA-4, CD80 and CD86, for T cell activation and inhibition has been established. In the previous study, we reported the results of a polymorphism screening of human CTLA-4 gene. In this study, we screened for polymorphisms of human CD28, CD80 and CD86 genes, and detected that polymorphisms were tested for the association with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Variations were identified in the coding regions of CD80 (452G/A, 614C/G and 864A/G) and CD86 (1057A/G), while no variation was observed in the coding region of CD28. The variations at CD80 position 452 and CD86 position 1057 were present in a substantial proportion of the Japanese population, and were considered to be single nucleotide polymorphisms within the coding sequence (cSNPs). CD80 864 (G-->A) leads to the amino acid substitution N186D, and CD86 1057 (A-->G) results in A304T substitution. Furthermore, in the analysis of CD80 5'-flanking region, six SNPs, -454C/A, -387T/C, -232G/A, -79G/C, -7T/C and /A, and one insertion, -558ins (CATGA), were identified. The combination of these variations was found to constitute four promoter alleles of CD80. None of the observed variations was significantly associated with RA or SLE. Further studies will be of particular interest to examine the functional difference of the promoter alleles for the transcriptional activity of CD80, as well as the evolutionary pathway of the four alleles.