Megakaryocytes from guinea pig bone marrow were isolated and maintained in liquid culture and were treated with ADP, thrombin, arachidonic acid, or collagen. Megakaryocytes spread with an active ruffled membrane in response to ADP (1-100 microM), thrombin (1.0 U/ml), and arachidonic acid (50 microM) but responded to collagen surfaces only if fibronectin was added to the cultures. Spreading could be blocked completely by dibutyryl cyclic AMP (dibutyryl cAMP) or isobutylmethylxanthine at 1 mM, as well as by cytochalasin D (2 microgram/ml), but not by colchicine up to 1 mg/ml. The distribution of contractile proteins was examined by immunofluorescence. In untreated, spherical cells, staining with antimyosin, antifilamin, anti-alpha-actinin, or with fluorescein-labeled subfragment 1 (FITC-S1) was diffuse and unpatterned. With antitubulin antibody, however, microtubules were seen in a dense array throughout the unspread cells. In actively ruffling spreading cells, myosin, filamin, and actin were visualized in the region of the ruffled membrane while alpha-actinin was seen most prominently in a band located proximal to the inner part of the ruffle. In fully spread cells, actin, myosin, filamin, and alpha-actinin were seen in filaments that filled the cytoplasm. Antimyosin and anti-alpha-actinin staining of the filaments was periodic with approximately 1 micrometer center-to-center spacing. Actin, filamin, and alpha-actinin were also identified in punctate spots throughout the spread cytoplasm. Microtubules were absent from the ruffle but filled the cytoplasm of fully spread cells. Rings, 1.5-2.5 micrometer in diameter, were seen with antitubulin in 13% of the spread cells. Our results show that megakaryocytes respond to platelet agonists, but typically by spreading, rather than extending, filopodia. From the changes in localization of contractile proteins and from time-lapse cinematography, we propose a model for cell spreading.