Abstract A subtractive hybridization approach was used to identify genes that are expressed at the beginning of gastrulation. We used tester DNA complimentary to RNA (cDNA) prepared from stages 6–7 embryos (gastrula) and excess driver cDNA from stages 2–4 embryos (syncytial blastoderm) to generate a gastrula-subtracted cDNA library. A reverse Northern blot procedure used to analyze 105 subtracted clones showed that 65% had a level of expression at least 2.5-fold higher in stages 6–7 versus stages 2–4 embryos. We determined the nucleotide sequence of these clones and identified 49 individual sequences, including 33 previously uncharacterized genes. We verified the level of expression of 24 genes during Drosophila melanogaster embryogenesis using a semiquantitative polymerase chain reaction (PCR) approach. As expected, all of the selected clones showed their highest level of expression after stages 2–4 of embryogenesis, including several that displayed peaks of expression during gastrulation. Three genes that were expressed at their highest levels in stages 6–7 were further analyzed by 5′-rapid amplification of cDNA ends (RACE) analysis, Northern blot assays and in situ hybridization. Our results indicate that these genes exhibited temporal and spatially restricted patterns of expression in developing embryos, and moreover, their transcripts were detected in cells that undergo morphological changes during the gastrulation stage. Characterizing the role of these genes will be important to increase our understanding of the molecular mechanisms that regulate cellular activities during D. melanogaster gastrulation.