Previous studies have shown that cells of the TE671/RD human clonal line express muscle-type nicotinic acetylcholine receptors (nAChR) and m3-type muscarinic acetylcholine receptors (mAChR) whose numbers and function are regulated by agonist treatment and second messenger modulation. Here we show that cytochalasin treatment, which causes disruption of actin networks, induces marked changes in the numbers and distribution of nAChR, but not mAChR. Moreover, whereas cytochalasin treatment fails to alter nAChR function significantly, it acutely potentiates mAChR-mediated phosphoinositide hydrolysis. Treatment of TE671/RD cells with different cytochalasin analogues (rank order efficacy at 5 micrograms/ml is H > J = B = C = D > A = E) produces a two- to fourfold increase in numbers of membrane-bound nAChR (Bmax in units of specific 125I-labeled alpha-bungarotoxin binding per milligram of membrane protein). nAChR up-regulation is evident after 1-2 days of cytochalasin B exposure, is maximal after 3-6 days of drug treatment, and is dominated by an approximately 10-fold increase (per cell) in an intracellular nAChR pool. Cytochalasin-induced nAChR up-regulation is similar in magnitude to, but not additive with, up-regulation of nAChR following chronic exposure to nicotine or phorbol ester. Northern blot analysis shows a four- to five-fold coordinate increase in levels of mRNA that encode nAChR alpha, beta, gamma, or delta subunits in cytochalasin-treated cells, suggesting that nAChR up-regulation has a possible transcriptional basis. Studies done using a 86Rb+ efflux assay indicate that cytochalasin treatment has no significant effect on nAChR function. By contrast, cytochalasin treatment has no effect on the numbers of mAChR as assessed by binding studies with the radioantagonist 3H-labeled quinuclidinyl benzilate, but it induces marked enhancement of carbachol-stimulated, but not basal, phosphoinositide hydrolysis. These studies suggest that presumed modulation by cytochalasin treatment of cytoskeletal microfilament integrity can differentially influence expression and function of mAChR (a prototype of the metabotropic receptor superfamily) and nAChR (a prototype of the ligand-gated ion-channel superfamily). The results also suggest possible new roles for the cytoskeleton in regulation of membrane receptor expression, function, and cross talk.