Objective: It is essential that the dental surgery autoclave be tested regularly to ensure that it is operating at a temperature and pressure that will kill all microorganisms. The aims of this study were to investigate the knowledge of general dental practitioners on aspects of autoclave use, to examine their autoclaving routines, and to test the effectiveness of the sterilization cycle of their practice's autoclave. Method and materials: A 35-question questionnaire was distributed to 840 dental practitioners in the United Kingdom. They were also sent three spore test ampules with details of their function and instructions on their use. The practitioners were asked to use the spore test ampules in their own practice's autoclave and to return these for testing. Results: Four hundred one questionnaires and associated spore test ampules were returned for analysis, a 47.7% response rate. Eight practice autoclaves (2.0%) failed to sterilize the spores. These practices were offered a retest following counseling. A total of 77.8% of respondents had received formal training in infection control. Of the 71.3% of respondents who stated that they check the performance of their autoclave routinely, 31.5% use a chemical strip every cycle, 17.5% use it once per day, and 30.4% use it once per week. However, only five respondents (1.2%) use a spore test; two of these respondents use it every cycle, one once per day, and two once per week. Conclusion: Further education of practitioners may be necessary to enhance their awareness of the need for routine checking of autoclave effectiveness.