The issues of cross-infection and the survival of variant Creutzfeldt Jakob disease (vCJD) on surgical instruments have highlighted the importance of cleanliness of multiple-use surgical instruments. The aim of this study was to assess the levels of total protein contamination on a wide range of surgical instruments as an indication of the effectiveness of routine cleaning and disinfection in hospitals. Anonymized trays of wrapped and autoclaved instruments were supplied to two laboratories for analysis at the stage where they would normally be returned to operating theatres. Instruments were assessed for residual protein and total organic matter. Laboratory A showed that 17% (35/206) of instruments were above a threshold that equated to 200 microg. The worst examples, a McIvor gag, a Draffin rod (child) and a Yankaur sucker, had 1.028, 1.286 and 2.228 mg of extractable protein, respectively. The median (25th, 75th percentiles) amount of protein from instruments from different hospitals assessed in Laboratory B ranged from 8 (3, 30)mug (Hospital C) to 91 (35, 213) mug (Hospital D) (P=0.044). The residual matter washed from instruments varied from 0.62 (0.32, 0.81) mg (Hospital E) to 3.5 (3.5, 4.0) mg (Hospital A) (P=0.0001). In one case, 45 mg of residual organic matter was washed from an instrument (split stem). In conclusion, this study demonstrated that a proportion of instruments at the point of use show levels of protein that could pose a direct cross-infection risk via prion agents and other organic contamination that may reduce the effectiveness of cleaning/disinfection strategies targeted against either prions or traditional infectious agents.