Movements of tissue hemocytes in the Eastern oyster Crassostrea virginica were monitored and quantified by image analysis of sections following inoculation with agar cores containing Escherichia coli or cell-free medium on which the bacteria had previously grown. Hemocytes respond to the presence of live bacteria by accumulating in widely dispersed areas of tissue surrounding the gut and digestive diverticula. The response is rapid and evident within 40 min, is maximal at 1 hr, and declines by 3 hr after inoculation. Sterile implanted agar cores do not produce a response. Bacteria killed with ozone elicit a response when inoculated together with the medium on which they had grown while bacteria killed by heat or formalin do not. Killed bacteria suspended in saline fail to stimulate hemocyte chemokinesis. Cell-free medium applied externally produces a response equal to that measured with live bacteria inoculated internally. Extraction of bacteria-free medium with hexane does not significantly reduce hemocyte chemokinesis. Digestion of bacteria-free medium with pronase completely eliminates chemokinesis. Molecular filtrates of bacteria-free medium induce maximal chemokinetic response at molecular weight as low as 1 kDa. These data show that the oyster hemocyte activators produced by E. coli are most likely low-molecular-weight polypeptides which diffuse from the site of inoculation and can pass through the intact external surface epithelium to induce a chemokinetic response.