The purpose of this investigation was to inspect and characterize the subgingival root surface after a 10-day exposure in vivo to 25% tetracycline hydrochloride by weight in an ethylene vinyl acetate copolymer fiber matrix with and without root planing therapy. The root surfaces were examined by fluorescent-light microscopy (FLM), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Thirty-two teeth were selected for study, 4 from each of 8 patients. The teeth of each patient were randomly assigned to one of four treatment groups: non-treated control (C), scaling and root planing only (RP), tetracycline-impregnated fiber only (F), and scaling and root planing with tetracycline-impregnated fiber application (RP/F). SEM revealed a visible reduction in the subgingival microbial flora in both the F and RP/F treatment groups in comparison with the C group specimens. Many of the residual bacteria observed in F and RP/F specimens appeared non-viable, exhibiting obvious loss of membrane integrity. In contrast, the RP specimens exhibited randomly distributed areas of residual subgingival plaque and calculus with newly developing plaque fronts; the plaque fronts undoubtedly having formed during the 10 days post-therapy. All RP/F specimens exhibited an incomplete removal of adsorbed root surface pellicle and demineralization of the subsequently exposed root surface. EDS analysis of large crystals adhering to root surfaces of F and RP/F specimens revealed high chloride peaks, suggesting the presence of residual tetracycline. FLM examination of F and RP/F treated specimens showed a superficial penetration of tetracycline into the root surface of about 10 microns. Areas of demineralized root showed slight tetracycline penetration into exposed dentinal tubules.