For tonsil B cells of a particular high density (below 65% Percoll), both phorbol myristate acetate (PMA) (5 ng/ml) and calcium ionophore A23187 (500 nM) were required to induce RNA synthesis, significant DNA synthesis also occurring in the presence of 12,000 MW B-cell growth factor (BCGF). In contrast, PMA alone, even at 1 ng/ml, was a sufficient stimulus to induce strong DNA synthesis in low-density B cells (45-50% Percoll) and strong proliferative responsiveness to BCGF in intermediate-density B cells (55-65% Percoll). In these latter B-cell populations, A23187 (500 nM), acted synergistically with non-mitogenic PMA doses to induce strong DNA synthesis, the PMA dose required being 5-50 times lower in low-density B cells (0.1-1 ng/ml) than in intermediate-density B cells (5 ng/ml). Preactivation for 30 hr with anti-Ig antibodies plus BCGF, known to drive B cells into late G1, rendered high-density B cells responsive to PMA (1-10 ng/ml) with high, dose-related DNA synthesis. These data indicate that the B-cell mitogenicity of a given nanomolar dose of PMA depends on the more advanced state of activation of B cells. It was also found that the above optimal dose of A23187 (500 nM) paradoxically inhibited the PMA-induced DNA synthesis of low-density B cells and in vitro preactivated high-density B cells. Data obtained with low-density B cells suggest that a calcium influx during the PMA-induced proliferative phase of B cells may provide a negative signal for the DNA synthesis.