In the surgical treatment of otosclerosis, the coupling between the stapes prosthesis and the long process of the incus is critical. After surgery, connective tissue and mucosa may grow over the coupling area and thereby influence the sound transmission properties of the incus-prosthesis interface. It was the hypothesis of this study that tissue ongrowth in the incus-prosthesis interface has little influence on sound transmission following stapes surgery. The goals of the study were to: (1) investigate the extent of postoperative tissue ongrowth over the stapes prosthesis; (2) objectively evaluate intra- and postoperative sound transmission properties of revision stapes surgery and compare the findings to those from primary surgery; (3) quantify the influence of ongrown tissue on sound transmission after stapes surgery. A group of 10 patients undergoing revision stapes surgery was investigated with audiological evaluations and intraoperative laser Doppler interferometry, and with scanning electron microscopy of the explanted incus with its adherent prosthesis in 6 patients. Results were compared to a group of patients undergoing primary otosclerosis surgery and temporal bone experiments. Results indicated that tissue grows over the prosthesis, as identified in all specimens. Sound transmission properties were evaluated intraoperatively (i.e., incus mobility and prosthesis-fixation quality), and found to correlate well with the functional hearing results. Ongrowing mucosa in the incus-prosthesis interface had only a minimal effect on sound transmission properties and cannot compensate adequately for insufficient prosthesis fixation. Therefore, it is essential that the stapes prosthesis is properly fixed during primary otosclerosis surgery.