The yeast Sir2 protein regulates epigenetic gene silencing and as a possible antiaging effect it suppresses recombination of rDNA. Studies involving cobB, a bacterial SIR2-like gene, have suggested it could encode a pyridine nucleotide transferase. Here five human sirtuin cDNAs are characterized. The SIRT1 sequence has the closest homology to the S. cerevisiae Sir2p. The SIRT4 and SIRT5 sirtuins more closely resemble prokaryotic sirtuin sequences. The five human sirtuins are widely expressed in fetal and adult tissues. Recombinant E. coli cobT and cobB proteins each showed a weak NAD-dependent mono-ADP-ribosyltransferase activity using 5,6-dimethylbenzimidazole as a substrate. Recombinant E. coli cobB and human SIRT2 sirtuin proteins were able to cause radioactivity to be transfered from [³²P]NAD to bovine serum albumin (BSA). When a conserved histidine within the human SIRT2 sirtuin was converted to a tyrosine, the mutant recombinant protein was unable to transfer radioactivity from [³²P]NAD to BSA. These results suggest that the sirtuins may function via mono-ADP-ribosylation of proteins.