We have initiated a biochemical analysis of factors that regulate the expression of the Ultrabithorax (Ubx) homeotic gene during embryogenesis. Transcriptionally active extracts have been prepared from Drosophila embryos at successive stages of development that recreate in vitro the temporal profile of Ubx gene expression during embryogenesis. Multiple sequence-specific transcription factors have been detected that bind to essential cis control elements located upstream and downstream of the Ubx mRNA cap site. The activity of some of these transcription factors varies during embryogenesis, and some are detected in embryonic extracts but not in Drosophila tissue culture cells. One factor, which binds to multiple GAGA DNA sequence motifs in the Ubx promoter, has been purified and shown to activate transcription from this promoter in a binding site-dependent manner. This in vitro analysis should help in understanding how Ubx expression is regulated and provide insight into the processes determining cellular fates during development.