Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying alpha-actinin from chicken gizzards was used to provide antigen for raising anti-alpha-actinin. Fluorescein isothiocyanate-labelled anti-alpha-actinin (FAalphaA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlongy gels against myosin, tropomyosin, actin, and alpha-actinin showed that antimyosin reacted only with myosin, anti-alpha-actinin only with alpha-actinin. Anti-alpha-actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAalphaA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAalphaA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of alpha-actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of alpha-actinin much easier to interpret.