Effects of ionophore A23187 on skeletal collagen formation were investigated in vitro. Collagen synthesis was quantitated in fetal rat calvaria by measuring [3H]proline incorporation into collagenase-digestible (CDP) and noncollagen protein (NCP) using purified bacterial collagenase; [3H]proline was added for the last 2 h of culture. Results are as follows. 1) A23187 (0.03–1.0 jug:/ml) inhibited incorporation of label into CDP and NCP after 24 h of culture, with a greater effect on CDP. The response was not associated with altered amino acid uptake, precursor pool size, or degradation of newly labeled protein. 2) Submaximal concentrations of A23187 and parathyroid hormone or dibutyryl cAMP decreased CDP formation to a greater extent than treatment with the agents alone. 3) Imidazole, while ineffective by itself, enhanced the effect of A23187. 4) Alteration of medium calcium did not affect the response to ionophore. 5) The inhibitory effect of A23187 was partially reversed by 24 h and completely reversed b...