The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. In order to enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kb promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of EGFP in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germline transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)(pA301) transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.