Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10-8 M. Inclusion of isobutylmethylxanthine decreased the concentration of PTH required to produce half-maximal stimulation to 2 ÃÂ— 10-10 M, indicating action via cAMP. With respect to the inhibitory activity, PTH and epidermal growth factor were the only agents, among those tested, able to enhance its production. Both hormones caused a 50âÂ€Â“100% increase over control levels 72 h after hormone administration. There were notable differences in the time courses of production of collagenase and the inhibitor. After treatment with PTH, the enzyme reached maximal concentrations between 12âÂ€Â“48 h, but declined to undetectable levels by 96 h. In contrast, the inhibitory activity was secreted in a linear fashion, with the highest concentrations achieved around 72âÂ€Â“96 h. These results suggest a complex pattern of regulation of collagenase and inhibitor secretion by the osteoblastic cell, with the steady accumulation of inhibitor perhaps being responsible for the ultimate curtailment of enzyme activity.