The 4 normal alleles of M1, M2, M3, and M4 are the most common gene products of the human alpha-1-antitrypsin (hAAT). Two single substitutions in M1 are responsible for M3 and M4, whereas 2 substitutions in M1 produce M2. Polymerase chain reaction-restriction fragment length polymorphism analysis of the Arg(101)/His(101) sequence variation can separate M1 and M3 from M2 and M4 alleles. To complete the genotyping procedure of hAAT M variants, the exon-V Glu(376)/Asp(376) sequence variation was directly analyzed using a designer primer with a single-base substitution in its sequence. This substitution induced an artificial site for the same restriction enzyme in the polymerase chain reaction product. The new restriction site was present in M1 and M4 but absent in M2 and M3, which can be applied as a rapid reliable means for the M-variant genotyping of hAAT.