The mechanism by which tetracyclines affect the formation of dentin was studied by measuring the biosynthesis of collagen in the pulp. The material consisted of 160 rabbit pulps and 108 rat pulps. Collagen synthesis was determined by incubating pulps with [14C]proline and measuring the formation of non-dialyzable [14C]hydroxyproline. The activity of protocollagen proline hydroxylase was measured as the conversion of [14C]proline to [14C]hydroxyproline in a protocollagen substrate by the supernatant of a pulp homogenate. In in vitro experiments, oxytetracycline or demethylchlortetracycline inhibited collagen synthesis. Also, the activity of protocollagen proline hydroxylase extracted from rabbit pulps was decreased in the presence of tetracyclines. In both cases the inhibition was related to the concentration of tetracyclines and the inhibition could be prevented by addition of ferrous iron to the incubation. In in vivo studies, injections of demethylchlortetracycline to rats inhibited collagen synthesis measured in vitro and the activity of protocollagen proline hydroxylase in the incisor pulps. It was concluded that this effect may be specific to collagen synthesis and the effect may be through chelation of ferrous iron, a cofactor of protocollagen proline hydroxylase. Consequently, the mineralization disturbances in developing teeth during tetracycline therapy may be partly due to a decreased formation of the organic matrix of dentin.