In this study alpha-1-antitrypsin (AAT) phenotypes at the protease inhibitor (PI) locus were determined by isoelectric focusing of native and desialylated serum samples from 236 Japanese subjects living in the western part of Japan. The shifts in relative mobility between some PI types were observed before and after desialylation. This technique was useful in distinguishing between some PI M subtypes and variants. The molecular basis of four variant alleles, including two new alleles found in this study, was characterized: PI E(tokyo) [Lys(335)(AAG)--> Glu(GAG)] and PI N(nagato) [Leu(276)(CTG)-->Pro(CCG)] arose from PI M1(Val(213)) and PI M2, respectively. A new PI P(yonago) [Asp(19)(GAT)-->Ala(GCT)] originated from PI M1(Val(213)). A new PI M5(gunma) [Pro(362)(CCC)-->Ser(TCC)], arising from PI M3, was the sixth allele involving a mutation at codon 362, which is suggested to be a mutation hot spot. PI M5(gunma) was likely to show normal AAT levels and function although the mutations occurred near codon 358 for Met(358). The molecular basis of PI variant alleles found in Japanese was different from that reported in previous studies.