The reuse of transgingival healing abutments has been advocated by several implant manufacturers, but cleaning and sterilization procedures to yield clean and optimal surfaces have yet to be developed. The objective of this in vitro project was to investigate various cleaning and sterilization regimens for the removal of biological debris to support reattachment of subgingival connective tissue. Simulated clinical healing abutment surfaces were exposed to culture medium with serum for 1 hour to simulate biological exposure. Simulated healing abutment surfaces not contaminated by serum were used to represent the "as-is" healing abutment surface without prior in vivo use. The discs were cleaned with detergent before sterilization by ultraviolet light (UV) or steam autoclaving (AC) both with and without 1- and 5-minute plasma cleaning (PC). A series of surface analytical techniques (XPS, AES, and surface contact angles) and in vitro analysis of cell attachment and spreading using gingival fibroblasts were performed. After exposure to the simulated biological conditions, clinical cleaning followed by UV resulted in contaminated surfaces and relatively high levels of cell attachment. PC before UV treatment enhanced surface energetics but did not affect cell attachment and spreading. AC increased surface wetting angles; which were decreased somewhat by previous PC. Cell attachment was significantly reduced by AC. Although some increase in cell attachment after longer plasma cleaning was noted in the AC group, no difference in cell spreading was seen in any AC group. Cell spreading seemed to be less for all AC groups compared with all UV, as-is, and control groups. Although certain cleaning (PC) and sterilization (UV) procedures can be effective for cleaning transgingival healing abutments, those using AC are questionable due to their propensity for organic and inorganic contamination and unfavorable surface alteration.