A biochemical assay employing DNase-I affinity chromatography, two- dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two- dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.