The secreted signalling molecule encoded by the wnt1 gene and the paired box-containing pax2 gene are thought to play an integral role in patterning the zebrafish rostral nervous system. Using a double-label analysis, we compare the expression patterns of wnt1 RNA and pax2 protein during zebrafish embryogenesis to determine whether they were expressed in identical or overlapping patterns in individual embryos. During gastrulation, wnt1 RNA was detected in a pattern similar but not identical to the pax2 protein. Later, wnt1 and pax2 co-localize to the midbrain-hindbrain boundary. Exogenous retinoic acid, a teratogen that is known to affect the formation of the midbrain-hindbrain boundary, has a profound affect on both wnt1 and pax2 expression at gastrulation. Furthermore, when pax2 is overexpressed in zebrafish embryos, the wnt1 pattern of expression expands ventrally in the prospective rostral neuroepithelium. Despite the widespread and random distribution of exogenous pax2 RNA, it alone is unable to induce wnt1 expression in other ectopic sites. These results are consistent with the coordinate expression of wnt1 and pax2 being in a pathway responsible for establishing the midbrain-hindbrain boundary and support the earlier interpretation that pax2 may regulate wnt1 expression [Krauss et al., 1992], although only in a subset of embryonic cells. These data suggest that a predisposition for the regionalization of the central nervous system exists at gastrulation.