BACKGROUND: There are no national guidelines specific for handling prion-associated specimens in Canadian medical laboratories. Medical laboratory workers may perceive themselves at risk of prion transmission and, on occasion, decline to process such specimens.OBJECTIVE: To examine the knowledge, attitudes and reported behaviours of medical laboratory workers in relation to prion disease to understand their risk perception and the need for national laboratory guidelines on prion infection control.DESIGN: Survey development and cross-sectional web-based administrationMETHODS: The survey was developed through key informant interviews and a modified Delphi process. Medical laboratory workers across Canada were invited by laboratory managers and national organizations to complete the web-based survey.RESULTS: Twelve key informant interviews were performed. Consensus for questionnaire content was reached through two rounds of the Delphi process. Responses were received from 426 Canadian medical laboratory workers; 37% of medical laboratory staff reported processing prion-associated specimens. Different protocols for specimen processing were followed, and 18% believed they were at risk when processing these specimens. Less than one-third of those receiving specimens believed they were adequately trained. The mean (±SD) knowledge score was 9.25±4.5/24; individuals who had received training scored significantly higher than those who were untrained (P<0.01). Eighty-one per cent of respondents would be more comfortable processing specimens if national guidelines existed and were used in their laboratory.CONCLUSION: There is a high perception of risk and few perceived benefits of processing prion-associated specimens. National guidelines for prion infection control in medical laboratories and adequate training would enable medical laboratory workers to process these specimens efficiently and confidently.