Factors affecting the sterility of the work area in barrier isolators and a biological safety cabinet (BSC) were studied. A study in a mother-and-child tertiary care teaching hospital in Canada examined the effects of a range of variables on the sterility of work areas in four barrier isolators and a standard BSC. The variables consisted of type of equipment (barrier isolator or BSC), day of the week (Monday through Thursday), time of day (0800-1000, 1000-1200, 1200-1400, and 1400-1600), sampling site (16 surfaces and 5 air sedimentation zones), type of product prepared (antimicrobial, total parenteral nutrient solution, etc.), cleaning procedure (before or after primary cleaning), and level of product preparation activity (none to intense). A total of 657 surface and air sedimentation samples, 327 plated onto Trypticase soy agar (TSA) and 330 onto Sabouraud dextrose agar (SAB-D), were taken during a 20-day period. Thirty-three (5%) of the samples yielded microbial growth when cultured (24 on TSA and 9 on SAB-D). A total of 74 isolates were identified, including Bacillus, Staphylococcus, Penicillium, Micrococcus, Corynebacterium, and Mucor species. Single-variable analysis showed that sampling site, sample type, the time of day samples were taken, and the types of equipment contributed significantly to microbial growth in the samples taken. Several variables were associated with microbial growth in samples from the work areas of barrier isolators and a BSC. More study is needed to compare BSCs and barrier isolators with respect to sterility.