During early developmental stages, the embryonic vertebrate brain is still relatively simple with few morphological landmarks that would indicate subdivisions in the prosencephalic primordium. To better understand the early organization of the rostral brain of a lower vertebrate, we investigated the embryonic development and regionalization of the fore- and midbrain of a small teleost, the zebrafish (Danio rerio). We used regulatory gene expression patterns to trace putative prosomeric domains to the beginning of the pharyngula period, when morphological manifestations of prosomeres are not immediately evident. We directly compared the expression domains of members of the dlx, emx, fgf, hh, lim, nkx, otx, pax, POU, winged helix and wnt regulatory gene families in the rostral brain by means of two-color whole-mount in situ hybridization. This allowed us to define precisely abutting expression borders of neighboring expression domains of different genes. Our analysis shows that the genes examined are expressed in anteroposteriorly and dorsoventrally restricted domains, and share expression borders at stereotypic positions within the fore- and midbrain. The arrangement of the various expression domains identified four major longitudinal subdivisions, which extend in parallel to the bent longitudinal rostral brain axis. Furthermore, we identified a series of eight transverse diencephalic domains which may indicate a prosomeric organization of the rostral zebrafish brain.