ABSTRACT As part of a large scale chemical mutagenesis screen of the zebrafish (Danio rerio) genome, we have identified 33 mutants with defects in hematopoiesis. Complementation analysis placed 32 of these mutants into 17 complementation groups. The allelism of the remaining 1 blood mutant is currently unresolved. We have categorized these blood mutants into four phenotypic classes based on analyses of whole embryos and isolated blood cells, as well as by in situ hybridization using the hematopoietic transcription factors GATA-1 and GATA-2. Embryos mutant for the gene moonshine have few if any proerythroblasts visible on the day circulation begins and normal erythroid cell differentiation is blocked as determined by staining for hemoglo-bin and GATA-1 expression. Mutations in five genes, chablis, frascati, merlot, retsina, thunderbird and two possibly unique mutations cause a progressive decrease in the number of blood cells during the first 5 days of development. Mutations in another seven genes, chardonnay, chianti, grenache, sauternes, weißherbst and zinfandel, and two additional mutations result in hypochromic blood cells which also decrease in number as development proceeds. Several of these mutants have immature cells in the circulation, indicating a block in normal erythroid development. The mutation in zinfandel is dominant, and 2-day old heterozygous carriers fail to express detectable levels of hemoglobin and have decreasing numbers of circulating cells during the first 5 days of development. Mutations in two genes, freixenet and yquem, result in the animals that are photosensitive with autofluorescent blood, similar to that found in the human congenital porphyrias. The collection of mutants presented here represent several steps required for normal erythropoiesis. The analysis of these mutants provides a powerful approach towards defining the molecular mechanisms involved in vertebrate hematopoietic development.