Elevations of intracellular cyclic AMP, achieved with the use of phosphodiesterase (PDE) inhibitors, cause functional downregulation of most inflammatory cells. Rolipram, an inhibitor selective for the PDE4 isozyme, can markedly downregulate antigen-driven proliferation and cytokine gene expression of unfractionated human peripheral blood mononuclear cells. However, it is unclear whether PDE4 inhibitors in a mixed-cell system exert their immunosuppressive effect on the lymphocyte or on the monocyte fraction. We have used an adherence-based protocol for separating peripheral blood mononuclear cells, isolated from atopic individuals, into lymphocyte and monocyte fractions and have selectively treated these populations with rolipram prior to reconstituting the cell cultures to their original lymphocyte/monocyte proportions. Cellular responses to both ragweed and tetanus toxoid were analyzed for both proliferation and gene expression of proinflammatory cytokines. A dose-dependent downregulation of ragweed- and tetanus toxoid-driven proliferative responses was achieved by pretreatment of lymphocytes from peripheral blood with rolipram. This downregulation was significantly greater than that achieved with pretreatment of monocytes. Pretreatment of both populations failed to show synergistic downregulation of proliferation. Lymphocyte pretreatment with rolipram also resulted in marked downregulation of gene expression for IL-4, IL-5, and interferon-gamma compared to monocyte pretreatment in both ragweed- and tetanus toxoid-driven systems. Interestingly, monocyte pretreatment in these systems resulted in significant downregulation of IL-2 gene expression compared to lymphocyte pretreatment. Flow cytometric analysis failed to show alterations in any of a panel of surface activation and signal transducing molecules by rolipram treatment with or without antigen stimulation. We conclude that, in a mixed cell system, PDE4 inhibitors downregulate antigen-driven proliferation and gene expression of proinflammatory cytokines predominantly through their effects on lymphocytes rather than monocytes.