Macrophages within the murine tunica muscularis were isolated and cultured for physiological studies. Following dispersion, macrophages were identified by phagocytotic activity of fluorescein isothiocyanate (FITC)-dextran. Immediately following isolation, macrophages were rounded and possessed fluorescent granula but developed a ramified shape after 3-4 days in culture. Resident and cultured macrophages were immunopositive for F4/80 and I-Ad/I-Ed. Greater than 90% of F4/80 positive cultured cells were FITC-dextran positive. Macrophages had resting membrane potentials (RMP) of -33.3 +/- 1.5 mV after 1 day in culture, which increased to -53.9 +/- 4.4 mV after 3-4 days. The change in RMP was associated with the development of an inward rectifying K+ current, and a decrease in a voltage-dependent, inactivating outward current. After 3-4 days in culture the inflammatory mediated substances adenosine triphosphate (ATP), platelet-activating factor and bacterial lipopolysaccharide induced increases in cytoplasmic Ca2+ ([Ca2+]i). Forskolin suppressed the ATP-induced increase in [Ca2+]i. Macrophages exhibited oxidative bursts, measured by oxidation of dihydrorhodamine-123 to rhodamine-123. Oxidative bursts coincided with a reduction in intracellular pH. Macrophages expressed a proton conductance that may participate in pH maintenance during reactive oxygen production. These results suggest that resident macrophages in the intestine may play a role in the immunological protection of the gut.