Two achaete-scute homolog sequences, Zash-1a and Zash-1b, were isolated from a zebrafish embryonic cDNA library. The Zash-1a cDNA encodes a protein very similar to rat Mash-1 and Xenopus Xash-1, with over 94% identity in the C-terminal three- fourths of all three polypeptides. The Zash-1b cDNA encodes a more distantly related protein, with 80% identity of amino acids to Mash-1 in this part of the sequence. At 24 hr, the Zash-1a transcripts are found in the hindbrain in two bilaterally symmetrical lines of cells which mark the boundary between the alar and basal plates and in rhombomere 1 in ventral cells near the floorplate. The gene is also expressed in particular regions of the telencephalon and diencephalon, in the epiphysis, the ventral tegmentum, the neural retina, and in specific cells in the spinal cord. Zash-1b transcripts are found in the hindbrain in segmentally arranged fan-like groups of cells which are located close to the anterior and posterior boundaries of each of rhombomeres 2-6 and in ventral cells close to the floor plate of most rhombomeres. The gene is also expressed at sites distinct from cells expressing Zash-1a in the tegmentum, diencephalon, telencephalon, and spinal cord. In the mutant cyclops, Zash-1a transcripts are absent from the ventral region of the tegmentum and in the ventral cells of rhombomere 1, while more dorsal expression regions are unaffected. The effects of the mutation on Zash-1b expression, however, are more complex. In the hindbrain, the ventral expression zone of this gene is absent, the more dorsal segmented expression is disorganized, and ectopic expression in the alar plate is observed. A dramatic ectopic expression is also observed in the anterior tegmentum. The cyclops gene, therefore, has both positive and negative effects on the CNS of the wild-type embryo: it is required for activation of both Zash-1a and -1b in particular ventral cells, but it also restricts the expression of Zash-1b in other ventral cells and in some dorsal regions. Zash-1a and -1b gene probes will be extremely useful in the analysis of additional mutations affecting development of the central nervous system in zebrafish embryos.