We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the M(r) of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 μg/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was no sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO.