Restriction map polymorphism in a 13-kb region of the Zw locus in Drosophila melanogaster was investigated for 64 X chromosome lines with seven 6-cutter and ten 4-cutter restriction enzymes. A total of 203 restriction sites were scored, of which 20 were found to be polymorphic. The estimated nucleotide variation for this region for overall data (pi = 0.003 and 0.001, and theta = 0.003 and 0.002, for 4-cutter and 6-cutter studies, respectively) was smaller than that reported for most regions studied in D. melanogaster. It was found that the Slow allozyme has a larger nucleotide variation and haplotype diversity than the Fast allozyme. Results suggest the relatively recent divergence of the Fast allozyme from the Slow allozyme. Glucose 6-phosphate dehydrogenase (G6PD) activity was measured as a phenotype of the Zw locus. A significant difference in G6PD activity between allozymes was detected. The between-line effect was highly significant within the Slow allozyme, but was not significant within the Fast allozyme. Although a direct causative link could not be established, these results suggest an association between the amounts of quantitative and molecular genetic variation at the Zw locus region.