Rolling represents the initial step leading to leukocyte extravasation from blood vessels during an inflammatory reaction. In vitro studies indicate that P-selectin could be one of the ligands on endothelium involved in the rolling phenomenon, although the molecular determinants responsible for this transient attachment in vivo are still undefined. Our objectives were to develop a blocking monoclonal antibody against canine P-selectin and to use it to investigate the role of P-selectin in leukocyte rolling in vivo using the technique of intravital microscopy. P-selectin was immunoaffinity purified from canine platelets and used for the production of monoclonal antibodies. One of the hybridomas generated, MD6, was shown by enzyme-linked immunosorbent assay and by flow cytometry to bind preferentially to stimulated platelets and to completely prevent binding of stimulated platelets to neutrophils. Visualization of canine mesenteric venules by intravital microscopy showed that administration of MD6 resulted in a marked inhibition in the number of rolling leukocytes (18.96 +/- 9.92 v 156.40 +/- 19.50 leukocytes/min, P < .05; 88.3% +/- 6.0% inhibition). Control antibody MD3 (which recognizes a nonfunctional epitope of canine P-selectin) had no effect on the number of rolling leukocytes or on their rolling velocity. These results show for the first time that P-selectin plays an essential role in leukocyte rolling in vivo, and therefore may be a key participant of the inflammatory response.