Following in vitro inoculation with C albicans or P aeruginosa, three impression materials - polysulfide rubber, irreversible hydrocolloid, and poly(vinyl siloxane) - were disinfected with either 0.1% or 0.02% chlorhexidine gluconate and a quantitative assessment of the microorganisms was made. Very few microorganisms were retained on the poly(vinyl siloxane) material immediately after microbial inoculation. The numbers of microorganisms on polysulfide impression materials diminished rapidly with time even without disinfection, and a 30-minute disinfection procedure resulted in total microbe elimination. With irreversible hydrocolloid, microorganisms persisted and disinfection procedures were considerably less effective. When the disinfection efficacy of three commercially available agents was compared using irreversible hydrocolloid impression material, chlorhexidine gluconate (0.2%) was found to be less effective than either glutaraldehyde (2%) or sodium hypochlorite (0.0125%); the latter two agents were comparable in their antimicrobial effect. Thus, a simple disinfection regime of 30 minutes with commonly available disinfectants may be effective in eliminating cross-contamination from impression materials.