Actin and myosin are associated with the platelet surface membrane and may be involved in platelet shape change and clot retraction. Previously we demonstrated that the adenine nucleotide affinity label 5’p-fluorosulfonylbenzoyl adenosine (5’FSOzBzAdo) can be incorporated into 200,000, 135,000, 100,000, and 43,000 molecular weight polypeptides in isolated platelet membranes but into only the 100,000 molecular weight membrane polypeptide using intact platelets. Because 5’FS02BzAdo can also be incorporated into the adenine nucleotide binding sites of rabbit skeletal muscle actin and myosin heavy chain, we used 5’FS02BzAdo to localize actin and myosin within human platelet membranes. Incubating unstimulated platelet-rich plasma, unstimulated gel-filtered platelets, or platelets previously stimulated with 10 PM ADP or 1 unit/ml of thrombin with 5’FS02Bz[3H]Ado labeled only the 100,000 molecular weight membrane polypeptide. However, incubating washed platelets with 5’FS02Bz[3H]Ado also labeled the 200,000 and 43,000 molecular weight polypeptides in a pattern similar to that of isolated membranes. Thus, the 200,000 and 43,000 molecular weight polypeptides are present on the cytoplasmic surface of the plasma membrane of resting and stimulated platelets but can be exposed on the membrane’s outer surface during the trauma of platelet preparation. The 200,000 molecular weight polypeptide was tentatively identified as myosin heavy chain. The 43,000 molecular weight polypeptide was shown to be actin by its interaction with DNase I. Labeled polypeptides of 100,000 and 120,000 molecular weight appeared to be associated with the 43,000 molecular weight polypeptide and, thus, could represent membrane actin attachment sites.