RFLPs were detected in the five subunit genes of the human muscle nicotinic acetylcholine receptor (nAChR) using genomic DNA or cDNA probes from the homologous mouse loci. The RFLPs at the alpha-, beta-, gamma-, delta-, and epsilon-subunit gene loci were analyzed for genetic linkage in 16 families (n = 188). Significant evidence was obtained for close linkage of the beta- and epsilon-nAChR genes and much greater genetic distance between the alpha-nAChR gene and the gamma/delta-nAChR gene complex. The linkage analysis program CRI-MAP was used to map the positions of the beta- and epsilon-nAChR genes relative to seven markers on chromosome 17. The results indicate the beta- and epsilon-nAChR genes are separated by about 5 cM and located in the region of chromosome 17p occupied by D17S1, D17S31, TP53, and D17S513. The statistical evidence was confirmed by hybridization of the beta- and epsilon-nAChR probes to a panel of human-hamster somatic cell hybrids. The alpha-, gamma-, and delta-nAChR genes were placed on a map of 13 chromosome 2 markers. The linkage analysis placed the nAChR genes at two sites on chromosome 2q about equidistant from the marker CRYGP1, with the alpha-nAChR gene about 27 cM proximal and the gamma/delta-nAChR gene complex about 31 cM distal to CRYGP1.