In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealis alpha 3B embryonic/larval skeletal actin gene, reside in a 156-nucleotide fragment of the promoter that spans nucleotides -197 to -42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box 3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box 1) exhibits two binding activities using bandshift analysis. One of these binding activities contains components antigenically related to a serum-response factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility-shift analysis. Although the shifted complex coelectrophoreses with the CArG box 1/serum-response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides -197 and -167. In the actin promoter, a downstream region can apparently fulfil this function.