Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore, in situ hybridization of mRNA is one of the most powerful and widely used -techniques in biology. Recent advances allow the reliable and simultaneous detection of mRNA transcripts, or combinations of mRNA and protein, in zebrafish embryos.Here we describe a standard protocol for visualizing the precise expression pattern of a single transcript or multiple gene products. The procedure employs fixation and permeabilization of embryos, followed by hybridization with tagged antisense riboprobes. Excess probes are then washed and hybrids are detected by enzyme-mediated immunohistochemistry utilizing either chromogenic or fluorescent substrates.