The biotin biosynthesis genes of Kurthia sp., which is an aerobic gram-positive bacterium, were cloned from Kurthia sp. 538-KA26 and characterized. Eleven biotin biosynthetic genes have been identified in Kurthia sp. Kurthia sp. has two genes coding for KAPA synthase, bioF and bioFII, and also has two genes coding for BioH protein, bioH and bioHII. In addition, three genes, orf1, orf2, and orf3, whose functions are unknown, were found in the biotin gene clusters of Kurthia sp. The bioA, bioD, and orf1 genes are arranged in a gene cluster in the order orf1bioDA, and the bioB, bioF, and orf2 genes are arranged in a gene cluster in the order orf2bioFB. These gene clusters proceed to both directions; the face to face promoters and two 40-bp of palindrome sequences exist upstream of the orf1 and orf2 genes. The bioC, bioFII, and bioHII genes are arranged in a gene cluster in the order bioFIIHIIC; a 40-bp of palindrome sequence exists upstream of the bioFII gene. The bioH and orf3 genes are arranged in a gene cluster in the order bioHorf3; a palindrome sequence was not found upstream of the bioH gene. These palindrome sequences are extremely similar to each other, suggesting that the orf1bioDA, orf2bioFB, and bioFIIHIIC gene clusters are regulated by biotin. Kurthia sp. does not have the bioW gene coding pimeloyl-CoA synthase, suggesting that pimeloyl-CoA may be produced by a different pathway than that of gram-positive bacterium B. subtilis or B. sphaericus, further suggesting a modified fatty acid synthesis pathway via acetyl-CoA instead as E. coli has.