The transcriptional regulation of a cytoskeletal actin gene during Xenopus laevis embryonic development has been investigated. New transcripts of this gene begin to accumulate at approximately the mid-gastrula stage, between 12 and 16 hours after fertilization, replenishing maternal supplies of this transcript. To study the molecular processes which act to determine the timing of transcriptional activation of this gene, a gene-injection assay was devised, utilizing a cloned copy of the gene which has been marked by a small DNA insertion. Accurately-initiated transcripts of the injected gene accumulate in concert with those of the endogenous gene, showing that injected genes can undergo developmental regulation. As little as 485 nucleotides of upstream sequence is sufficient for proper temporal control of activation of an injected gene. The results presented here demonstrate the feasibility of a microinjection assay for the identification of regulatory gene sequences and transacting regulatory factors in amphibian embryos. Such an assay will be useful in achieving an understanding of general transcriptional control mechanisms acting in early development, and should also provide a means to study certain aspects of long-standing developmental problems, such as cytoplasmic localization and embryonic induction.