We have investigated the actin-related sequences in soybean using heterologous actin DNA probes from Dictyostelium, Drosophila, and yeast. Southern blot analysis of restriction digests of soybean DNA indicates that actin is encoded in a small multigene family. In order to isolate individual members of this gene family, we have constructed a soybean genomic library in the lambda vehicle Charon 4A. A partial characterization of this library shows it to be nearly complete. We have isolated from this library a number of recombinant clones that hybridize to actin-coding sequences from all three heterologous probes. We have identified the fragments containing the actin-related sequences on the physical maps of two of these clones lambda SAc1 and lambda SAc3. These fragments were subcloned in the plasmid vehicle pBR322. Using electron microscope heteroduplex mapping we show that the subclones, pSAc1 and pSAc3, share homology with the entire actin-coding sequence (1.1 kb) of Drosophila and Dictyostelium. Furthermore, pSAc1 and pSAc3 have additional homology of approximately 0.22 kb at the 5' ends of their coding sequences. No homology is detected in the 3' flanking regions of these clones. The actin sequence in pSAc1 contains an interruption of approximately 0.30 kb located 0.39 kb from the 5' end of the actin polypeptide coding region.