Abstract: When vinyl isolators and breeding rooms are used for microorganism control, it is important to identify any weak spots in the walls promptly to ensure continuous airtightness.We aim to establish a method of locating possible weak spots in the walls of vinyl isolators and breeding rooms.Wind speed, air pressure, temperature and humidity were measured in various areas within a vinyl isolator under each of five pressurization conditions induced with a blower.The areas adjacent to the weakest points in the walls were identified based on differences between expected values and the actual measurements in those areas.The maximum margin of error between the estimated and actual areas of weakness was 18%.We judged that this margin of error was within the range of normal prediction.In a breeding room, we estimated areas of weakness by the same method using an instrument that measures airtightness, and were able to identify the areas of weakness easily.We expect that the measurement methods established in this study will be useful in controlling airtightness in vinyl isolators and breeding rooms.Our object is to establish a leak test enabling us to estimate the sizes of leaks in vinyl isolators.Several vinyl isolators composed of vinyl parts (vinyl cap, sterile lock and sleeve) and with chamber size 115 × 50 × 45 cm were prepared for this study.An artificial hole with a diameter of 0.1, 0.2, 0.3, 0.5, 0.6, or 1.0 mm was made in each vinyl isolator, and the vinyl isolators were pressurized with blowers.An additional chamber without a hole was used as a negative control.The proposed leak test was then performed as follows.The vinyl isolators were pressurized to either 100 or 300 Pa.Temperature, humidity, and air pressure changes in the vinyl isolators with holes were simultaneously measured using a Testo-435 air quality measuring instrument.The severity of the decrease in air pressure resulting from a leak was proportionate to the size of the hole.Furthermore, the final air pressure value depended on the initial pressurization (100 or 300 Pa) induced by the blower.These results suggest that this leak test is a useful means of estimating leak size in vinyl isolators.