Peptidylarginine deiminases (PADs) convert arginine residues in proteins into citrullines. They are suspected to be involved in multiple sclerosis and rheumatoid arthritis pathophysiology, and they play a role in epidermis homeostasis and possibly in regulation of gene expression through histone modification. In humans, four isoforms encoded by the genes PADI1–4 are known so far. We here report the characterization and comparative analysis of the human (355 kb) and mouse (240 kb) PAD gene clusters on chromosomes 1p35–36 and 4E1, respectively. We characterized an as yet unknown human PADI6 gene, and cloned the corresponding cDNA encoding a 694-amino-acid protein. RT-PCR analysis showed a rather restricted pattern of tissue-specific expression, mainly in ovary, testis and peripheral blood leukocytes. Nucleotide substitution rates suggest that PADI genes are under purifying selection. Comparative analysis of the human and mouse sequences identified 251 conserved non-coding segments predominantly clustered within the promoter regions, the large (>10 kb) first intron of each of the genes PADI1–3, and an 8 kb PADI1–2 intergenic region. The presence of numerous transcription factor binding sites suggests the segments are putative regulatory elements. This study is the first description of the human PADI6 gene and encoded protein, and the first step towards a better understanding of the coordinated regulation of PADI gene expression.