Analysis of isozyme variability in four natural populations of Anopheles quadrimaculatus Species A indicated that the loci for Malic enzyme (Me) and Mannose phosphate isomerase-1 (Mpi-1) are on the X chromosome. There were female heterozygotes, but no male heterozygotes were observed. Strains fixed for fast- and slow-migrating allozymes were devised and crossed. Progeny phenotypes conformed to expectations for sex linkage; female progeny were heterozygous, and male progeny were hemizygous for the maternal allele. The three-point cross, using the Me and Mpi-1 loci with the sex-linked mutant rose eye (ro), established the gene sequence Mpi-1-11.1-Me-40.8-ro.