were further categorized into the percentages of phagocytic PAM's containing one (27 percent), two (15 percent), three (11 percent), four (8 percent), five (7 percent), and more than five (32 percent) spheres. The average percentage of cells with one or more spheres measured on 41 normal rats was 28.2 ± 8.2 percent.Since this percentage was lower than anticipated, we designed experiments to determine if all macrophages in the alveolar region were being exposed to spheres and if the spheres were in high enough concentration. Rats were again instilled in vivo with green fluorescent spheres for 2 hours. Samples were washed to remove nonphagocytized spheres and then incubated in vitro for 30 minutes with red fluorescent spheres 1.65 ,um in diameter (7). Microscopic examination showed that 35 percent of the PAM's had both red and green spheres, more than 60 percent contained only red spheres, and less than 5 percent had no spheres. Thus there were PAM's within the lungs that had not phagocytized green spheres but were capable of phagocytizing red spheres in vitro. These results suggested that the instillation of 1 x 107 to 2 x 107 spheres was insufficient to distribute them to all macrophages within the lungs. To alleviate this problem, we killed the rats, surgically exposed the trachea, opened the thoracic cavity, and instilled 15 ml of green fluorescent spheres (5 x I0 per milliliter) into the lungs hydrostatically (25 cm H20) by way of the trachea. After 30 minutes the lungs were lavaged and the percentage of phagocytic cells was quantitated microscopically and by FCM analysis. The results showed that more than 90 percent of the cells were phagocytic.Finally, to determine if microspheres were being phagocytized or instead were being nonspecifically bound to the cell surface, rat PAM's were exposed in vitro to 0, 0.1, 0.3, and 1.0 percent sodium azide and green fluorescent spheres. Experimental FCM data showed ( Table 1) that 91 percent of PAM's that were not exposed to sodium azide had associated spheres, whereas only 6 to 7 percent had spheres associated with them when exposed to 0.3 to 1.0 percent sodium azide. The microscopically determined phagocytoses were within 5 percent of the FCM results. Decreased phagocytosis, induced by sodium azide, was also shown in terms of a reduction in the number of particles per cell. Thus, as determined by FCM, the percentage of PAM's containing one to five spheres increased, whereas the percentage con-taining more than five spheres decreased. These data show that, as the inhibitor concentration increased, the percentage of phagocytic PAM's decreased and those PAM's that were phagocytic had fewer spheres.Although Sprague-Dawley rats were used in these studies, results may differ among rat strains and animal species. Since the microspheres contain surface carboxyl groups, they can be coated with opsonins, antibodies, or other chemicals for studying specific receptor-mediated phagocytosis (8). This technology has broad application for the rapid and accurate determination of phagoc...