O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.