BACKGROUND. Recommendations in the literature conflict on the necessity of disinfecting single-use vials prior to aspiration of fluid. Interventions to disinfect the stopper surface on multiple-dose vials vary considerably. OBJECTIVES. To determine the necessity of alcohol disinfection of the stopper on single-dose vials and to compare povidone-iodine and alcohol versus alcohol-only disinfection of the stopper prior to each needle penetration on multiple-dose vials. METHODS. The rubber stopper surfaces of 100 single-dose vials were cultured for the presence of bacteria. To determine the efficacy of two procedures for disinfection of multiple-dose vials, 87 stopper surfaces routinely disinfected with both povidone-iodine and alcohol were cultured for bacteria. After a change in practice, 100 multiple-dose vials routinely disinfected with alcohol only were cultured for the presence of bacteria. RESULTS. Of the cultures done on single-dose vial stoppers, 99% were sterile. A comparison of the two disinfection techniques for multiple-dose vials revealed that 83 (95%) of the 87 vials prepped with both povidone-iodine and alcohol were sterile, compared with all stoppers disinfected with alcohol only. CONCLUSIONS. This study shows the lack of necessity of any disinfection procedure on the rubber stopper of single-dose vials and the efficacy of alcohol only for disinfecting the stopper of multiple-dose vials.