We have recently described a sequential salt/ double-detergent technique for the separation of three subcellular fractions; cytosol [Nonidet P-40 (NP-40; RDH, Poole, Dorset)-soluble], a fraction enriched in cytoskeletal components (1 30 mv-KCI extract) and a membrane fraction [Triton X-100/ deoxycholate (DOC)-soluble] [ 11. These fractions contained free, cytoskeletal-bound and membranebound polysomes respectively [ 1, 21. Compelling evidence suggests that a large proportion of cytoskeletal-bound polysomes are associated with the microfilament system of the cytoskeleton (reviewed in [3]). The amount of cytoskeletal-bound polysomes in a cell may thus be dependent on the integrity of microfilaments. The regulation of the length and complexity of actin-containing microfilaments is governed by a family of actin-binding proteins (ARPs) [4]. The polymerization of actin, which occurs, for example, early after insulin stimulation [5, 61, is thus likely to be imparted by a change in the state of activation or subcellular distribution of this group of proteins. Experiments were designed to identify ARPs in extracts obtained after the application of the sequential salt/double-detergent technique [ 1 ] to the plasma membrane/mitochondria (PM), endoplasmic reticulum (ER) and nuclear (N) fractions prepared by disruption of Krebs I1 ascites cells using nitrogen cavitation [7]. A modified immunoblotting procedure was developed to identify ABPs. The method was briefly as follows: proteins were electrophoretically transferred to nitrocellulose membranes following SDS/PAGE of extracts, membranes were then pre-incubated for 30 min at 37°C with a 3% (w/v) solution of albumin in PRS (phosphate-buffered saline). After washing with PRS containing 0.05% Tween (PRS-Tween), incubation was performed with actin (5 pg/ml in PRS) for 30 min at 37°C to allow interaction with AHPs bound to the nitrocellulose membrane. Following washes with PRS-Tween, monoclonal anti-actin (Amersham International, Amersham, Bucks, U.K.), diluted 1 : 10000 in PRS containing 3%