Overlapping genomic clones of the human alpha 2-macroglobulin (alpha 2M) gene were isolated from a cosmid library and were used to map 80 kb of the chromosomal region of this gene. Fragments carrying the two exons encoding the bait region and the exon encoding the thiolester site were partially sequenced and PCR primers were designed for the amplification of both functional domains. By direct genomic sequencing of these domains in 30 healthy individuals and in 30 patients with chronic lung disease three mutations were detected. The first was a sequence polymorphism occurring near the thiolester site of the gene, changing Val1000 (GTC) to Ile1000 (ATC), with allele frequencies of 0.30 (GTC) and 0.70 (ATC), respectively. No difference of alpha 2M serum levels was observed for these two alleles. The second mutation occurred within the thiolester site of one patient, changing Cys972(TGT) to Tyr972(TAT). Since activation of the internal thiolester formed between Cys972 and Gln975 in each of the subunits of the tetrameric alpha 2M is involved in the covalent cross-linking of the activating proteinase, this mutation is predicted to interfere with alpha 2M function. The alpha 2M serum level was within the normal range in this patient. In one healthy individual we detected an alteration of the bait region sequence, which is usually encoded by two different exons separated by an intron of size 1.6 kb. In this individual, PCR amplification of genomic DNA using the bait region primers produced the common fragment of size 1.8 kb and an additional variant fragment of size 0.23 kb. This finding, and the genomic sequencing data of this individual, indicate that he carries two different alleles of the alpha 2M gene: one with the regular structure (bait exon I-intron-bait exon II), the other with the two bait exons fused into one. Direct genomic sequencing of the two alpha 2M functional domains is a useful tool for the detection of the genetic, and possibly the functional, heterogeneity of alpha 2M. This, in turn, may provide some insight into the hitherto unknown physiological role(s) of alpha 2M, by studying in vivo effects of naturally occurring mutations of the gene.