Human neutral alpha-glucosidase C (GANC) can be separated from the homologous mouse isozyme by starch gel electrophoresis at pH 6.5. A total of 40 clones (13 primary and 27 secondary) were derived from eight separate hybridization experiments between the mouse HPRT deficient RAG cell line and eight different human long term lymphoid cell lines or fetal cells. The thirteen primary clones showed 100% concordance between the expression of the human enzyme and the presence or absence of human chromosome 15. Analysis of the 27 secondary clones showed only two subclones discordant for segregation of human GANC and enzyme markers for 15. The two apparently discordant clones for human GANC were both derived from the same RAG X human fetal lung primary clone, and both lacked GANC activity, while retaining a 15. Since human GANC is polymorphic with a null allele at high frequency (MARTINIUK and HIRSCHHORN, 1980), it is possible that these subclones carried one chromosome with a null allele for GANC. Alternatively there could been an undetected chromosome break between the GANC locus and the loci of the marker enzymes. Whatever the reason for the two apparently discordant subclones, combined data from all 40 clones show 95% concordant segregation for human GANC and No. 15.