The regional gene assignments for human porphobilinogen deaminase (PBGD; EC 220.127.116.11) and esterase A4 ESA4; EC18.104.22.168) on chromosome 11 have been determined with somatic cell hybridization and immunologic, electrophoretic, and cytogenetic techniques. Dimethyl sulfoxide-induced erythroid differentiation of hybrid clones derived from the fusion of tetraploid Friend murine erythroleukemia (2S MEL) cells deficient in thymidine kinase and human Lesch-Nyhan fibroblasts (HLN) deficient in hypoxanthine phosphoribosyltransferase (HPRT-; EC 22.214.171.124) were examined for expression of human PBGD, ESA4, and lactate dehydrogenase A (LDHA; EC 126.96.36.199). Human PBGD was detected by rocket immunoelectrophoresis with rabbit anti-human PBGD IgG and by isoelectric focusing. The human chromsome complement of each clone was determined by cytogenetic and enzyme marker analyses. Of the five primary 2S MEL-HLN clones examined, three were positive for human PBGD, These were subcloned to yield a total of 10 secondary, tertiary, or quaternary clones. Analyses of these subclones permitted the regional assignment of human PBGD and ESA4 to the long arm of chromosome 11. Finer regional assignment of the loci for human PBGD and ESA4 was facilitated when two 2S MEL (HPRT-)-human fibroblast (HX/11) hybrids, each containing the X chromosome-autosome translocation (der 11), t(X;11)(q25-26;q23) as the only human chromosome, were examined for expression of human PBGD, ESA4, and LDHA. One clone, HX/11-2, contained the intact X/11 translocated chromosome; in the other, HX/11-3, 11p was deleted, and the human X/11 derivative was translocated onto a mouse chromosome. HX/11-2 expressed human LDHA, but HX/11-3 did not, verifying that the latter human 11/X derivative did not include 11pter→11p12; PBGD and ESA4 were not detected in either hybrid. These results confirm the location of the gene for human PBGD on chromosome 11 and establish the assignment of the loci for PBGD and ESA4 in the region 11q23 →11qter.