The primary sex determination signal, the X chromosome-to-autosome (X/A) ratio, controls the choice of sexual identity in the Drosophila melanogaster embryo by regulating the activity of the early promoter of the Sex-lethal gene, Sxl-Pe. This promoter is activated in females (2X/2A), while it remains off in males (1X/2A). Promoter activation in females is dependent upon X-linked numerator genes. One of these genes, sisterless-b (sis-b), corresponds to the scute (sc) locus of the achaete-scute complex, and it encodes a helix-loop-helix transcription factor. In the studies reported here we have used monoclonal antibodies to study the expression and functioning of the sc(sis-b) protein. Sc is first detected at nuclear cycle 6 to 7, well before Sxl-Pe is first active. At this stage, the protein is in the cytoplasm, not the nucleus. Only after the formation of the syncytial blastoderm, at nuclear cycle 10 to 11, does a substantial fraction of the protein enter the nucleus, and this nuclear import closely coincides with the initial activation of Sxl-Pe. Consistent with the idea that the dose of sc(sis-b) is critical for its function as an X-chromosome counting element, wild-type syncytial blastoderm embryos could be grouped into two classes based on the level of protein. Western blot (immunoblot) analysis demonstrates that this difference in protein level correlates directly with the activity state of the Sxl gene. Finally, we provide the first direct evidence that Sc forms heteromeric complexes in vivo in early embryos with the maternally derived helix-loop-helix protein Daughterless. This in vivo complex is likely to be critical for Sc function in Sxl-Pe activation.