Hybrid males of Chironomus thummi piger female x Ch. th. thummi male crosses were backcrossed with females of both parental stocks. Fourth-instar larvae of these backcrosses showed sex specific differences in the pairing behavior of region D3d-g in chromosome arm F of salivary gland chromosome III. Analysis of the banding pattern of region D3d-g after RB and quinacrine staining demonstrated that in piger female x thummi male hybrid males a single selectively stained band occurs within this region in the heterozygous condition at map position D3e1. This band could only be found in the thummi chromosome partner, it is heterochromatic and contains AT-rich DNA. In female hybrid larvae, however, such a selectively stained band is present in neither the thummi nor the piger chromosome region D3d-g. From these results it is concluded that the selectively stained band D3e1 represents the male sex determiner of our Ch. th. thummi stock and that the male is the heterogametic sex.