Adequate blood culture volume is the single most important determinant for yield of organisms. We sought to determine whether health care professionals are aware of the current evidence-based recommendations for blood culture collection. An anonymous survey of employees qualified to collect blood cultures was conducted (July-October 2004) in an urban tertiary care facility. The survey asked,"What volume of blood should be collected in one bottle for a blood culture?" Of the 360 employees of the hospital surveyed, 355 returned evaluable answers for blood culture volume collected. Overall, 79% (95% confidence interval [CI], 74%-83%) answered less than 10 mL, and 44% (95% CI, 39%-49%) answered less than 5 mL of blood. When examined by occupation, 90% (95% CI, 86%-94%) of nurses, 97% (95% CI, 91%-100%) of technicians, and 55% (95% CI, 46%-64%) of physicians answered less than 10 mL; 52% (95% CI, 45%-59%) of nurses, 63% (95% CI, 46%-79%) of technicians, and 26% (95% CI, 18%-35%) of physicians answered less than 5 mL. Of all respondents, 21% (95% CI, 17%-25%) answered 1 mL or less. Our findings reveal that a high percentage of health care personnel do not know the optimal volume of blood recommended for collection. Because volume remains the most important determinant for the optimal yield of organisms, these findings raise an important quality assurance issue.