The in vivo distribution of tetracycline in canine bone was studied by determining the location and intensity of tetracycline fluorescence in undemineralized sections of bone from multiple skeletal sites in fifteen animals which received varying doses of the different tetracyclines. The tetracycline fluorescence was compared with mineral content, as revealed by microradiography and, in one animal, with the in vivo distribution of Ca⁴⁵ as determined by autoradiography.

The tetracyclines were incorporated into every surface that was undergoing active deposition of new bone and they remained there indefinitely. Resorption cavities and inactive surfaces showed tetracycline fluorescence immediately after an intravenous dose, but this fluorescence rapidly disappeared. The tetracyclines were also incorporated into the skeleton in three other ways which were, probably, not related to new-bone formation:

1. Tetracycline was firmly and diffusely bound at low concentrations to areas that were not sites of the elaboration and mineralization of new matrix. This diffuse component paralleled closely the diffuse distribution of Ca⁴⁵ and appeared to follow the calcium distribution pattern of secondary mineralization and long-term exchange.

2. Tetracycline fluorescence was seen surrounding many lacunae. This may be either an additional form of tetracycline incorporation into bone or an artefact.

3. Tetracycline was also incorporated into the surface of many systems, which were interpreted as non-growing systems, in conjunction with the process termed edge sclerosis.

The presence of tetracycline incorporation into the skeleton by means which are not related to new-bone growth makes it impossible to estimate skeletal accretion accurately from a chemical determination of the total tetracycline content of a bone sample.

The extreme non-homogeneity of the distribution of active new-bone growth sites throughout the skeleton was impressive. This means that an interpretation of metabolic activity based on observations made on a limited sample is very hazardous.

The tetracyclines, however, do mark all sites of active new-bone formation and can be used as reliable intravital stains in properly designed and properly controlled experiments for the accurate determination of rates of accretion of bone.