The in vitro migration inhibition responses of peripheral blood leukocytes from tuberculin skin test positive and negative donors were tested to validate and determine optimal conditions for the agarose droplet method in the human. In vitro migration inhibition was observed in skin test positive donors in the presence of 25 microgram PPD/ml of medium using both unfractionated leukocytes and a mixture of immune lymphocytes with allogeneic polymorphonuclear leukocyte indicators. The supernatants of tuberculin positive lymphocytes cultured with PPD also inhibited the migration of human polymorphonuclear leukocytes and guinea pig peritoneal exudate cells but did not alter that of murine peritoneal exudate cells. These studies establish that the agarose droplet method is an efficient approach to the in vitro assessment of cell-mediated immunity in the human and define suitable indicator cell populations for assay of human lymphokines.