The gene encoding the neural cell adhesion molecule, N-CAM, has been localized on mouse chromosome 9. A BALB/cJ mouse genomic library prepared in lambda bacteriophage EMBL4 was screened by using a cDNA probe, pEC204, that corresponds to the coding region of the chicken N-CAM gene. Four weakly reactive and one strongly reactive recombinant phage were isolated. A region of the latter that was strongly homologous to pEC204 was subcloned to yield a new probe, pEC501. RNA transfer blots and nucleotide sequencing indicated that pEC501 encoded part of the mouse N-CAM gene. This probe defined a unique genetic locus, Ncam, associated with a restriction fragment length polymorphism that allowed the definition of two alleles. The locus could be provisionally assigned either to chromosome 9 or to chromosome 10 by correlating the presence or absence of mouse-specific DNA fragments reactive with the probe in a panel of somatic hybrid cell lines with the presence or absence of the various mouse chromosomes. Analysis of the inheritance of the Ncam-associated DNA polymorphism in recombinant inbred strains of mice revealed close linkage between Ncam and the Lap-1, Sep-1, and Thy-1 loci on chromosome 9. This result suggests an additional linkage between Ncam and the locus for the cerebellar mutation staggerer (sg). The Ncam locus provides an important reference point for mapping the genes for additional cell adhesion molecules as well as genes for other molecules involved in neural development and function.