Two neurological mutants deafwaddler (dfw) and opisthotonos (opt) and a cluster of three Shaker-like potassium (K) channel genes Kcna1, Kcna5, and Kcna6 were all independently mapped to distal mouse chromosome six (Chr 6). In this study, genetic and molecular techniques were employed to assess directly the linkage of the two mutants and to investigate the likelihood that a mutation in one of the three K channel genes may underlie dfw and/or opt. Genetic crosses testing for allelism showed that the dfw and opt mutations complement each other. Additional crosses demonstrated that the mutants are separated by a recombination distance of 3.1 +/- 1.8 cM. Microsatellite marker analysis of the crossover chromosomes recovered from the opt, dfw recombination study indicated that opt maps centromeric to dfw. The location of the K channel genes relative to the dfw mutation was determined by mapping these genes and 15 microsatellite markers in an intersubspecific backcross (IB) segregating for dfw [(CAST/Ei-+/+ x C3HeB/FeJ-dfw/dfw) x C3HeB/FeJ-dfw/dfw]. Analysis of the backcross progeny positioned the dfw locus in the interval between the microsatellite markers D6Mit11 and D6Mit55, D6Mit63. The K channel cluster maps telomeric to dfw. This study establishes the gene order cen-opt-dfw-Rho (D6Mit44)-Kcna1, Kcna5, Kcna6 on distal mouse Chr 6 and suggests that the neurological mutants opt and dfw affect two different genes, neither of which is caused by a mutation in any one of the three clustered K channels.