Cytoskeletal actin genes undergo developmentally timed transcriptional activation at the gastrula stage of embryonic development in the amphibian Xenopus laevis. To study the regulation of this process, a molecularly marked cloned actin gene has been introduced into living embryos by microinjection, and levels of its transcripts (which are distinct from endogenous actin message) have been measured by RNase protection. In vitro mutagenesis of the marked gene, followed by microinjection and transcriptional analysis of various mutants, has been used to search for gene sequences that participate in accurate transcriptional initiation and developmental control. Deletion mutants containing only 90 nucleotides of upstream sequence undergo correct developmental regulation, while deletion to -33 prevents normal activation of the gene. In the presence of sufficient upstream sequence, an actin-globin fusion gene, containing only 564 nucleotides downstream of the actin gene transcription startsite, is correctly activated. Taken together, these results imply that all sequences necessary for correct temporal regulation reside between -90 and +564 nucleotides, with respect to the transcriptional start site of the actin gene. They further suggest that developmental activation of actin gene transcription may involve either (1) interaction of non-DNA binding proteins with basal transcription factors, or (2) the concerted action of ubiquitous promoter-binding factors and factors that interact with downstream regulatory regions.