Normal and tumor-bearing host (TBH) peritoneal macrophage (M phi) responses to in vitro lipopolysaccharide (LPS) treatment were measured by assessing functional and phenotypic changes. Both normal and TBH untreated M phi suppressed mixed lymphocyte reaction (MLR) reactivity at all concentrations. Normal host M phi treated with LPS for 3 h were suppressive at all concentrations. TBH M phi treated with LPS for 3 h were not suppressive in the MLR until more than 5% were added. Surprisingly, 24 h treatment of normal and TBH M phi with LPS induced cells that significantly enhanced MLR reactivity when added at 2% or 5%. These cells were not suppressive until a 20% M phi concentration was reached. LPS treatment of normal and TBH M phi changed the percentage of cells expressing the surface markers Mac-1, -2, -3, and Ia as determined by flow cytometry. Normal host peritoneal M phi treated with LPS for 3 h had decreased Mac-1 and -3 expression, but there was no change in Mac-2 or Ia. Plating for 24 h did not change the percentage of M phi expressing Mac-1, -3, or Ia but did cause an increase in Mac-2+ M phi. Treatment of normal host M phi with LPS for 24 h led to a decrease in Mac-1+ and Ia+ M phi, no change in Mac-3+ M phi, but an increase in Mac-2+ M phi. LPS treatment of TBH M phi for 3 h decreased the number of Mac-1+ M phi, but Mac-2+, -3+, or Ia+ M phi numbers did not change. Plating TBH M phi for 24 h caused a decrease in the number of Mac-1+ M phi, no change in Mac-3+ or Ia+ M phi, but an increase in Mac-2+ M phi. Treatment with LPS for 24 h led to no change in the number of Mac-1+, -3+, or Ia+ TBH M phi, but Mac-2+ M phi increased. The phenotypic and functional changes after LPS treatment led us to ask if these changes were detectable at the level of DNA and RNA. Flow cytometric analysis of acridine orange-stained M phi was used to measure DNA and RNA levels. This analysis determines M phi cell-cycle kinetics and estimates their RNA synthesis. In normal host M phi, a 3-h LPS treatment caused a decrease of cells in G0/G1 but an insignificant change in RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)