Effects of metabolic gas accumulation on termite behavior in tightly sealed laboratory bioassay arenas were compared with arenas with partial air exchange, or open ventilation. Laboratory studies with termites are often conducted in glass or plastic arenas. These studies can last from a few days up to 12-16 weeks or longer. Based on personal observation and published laboratory studies, how tightly study arenas are sealed is not consistent. In some instances arenas are tightly sealed with impermeable rubber stoppers or tight-fitting lids. In other studies, arenas are covered with semi-air-permeable Parafilm®, or loose-fitting aluminum foil or ventilated lids to allow air exchange. Generally, arenas are covered and not completely open to the air, thereby maintaining high moisture requirements needed to avoid termite dehydration. In this study, to determine metabolic gas accumulation and gas effects on termite groups collected from two field colonies, bioassay arenas were capped with either tightly sealed metal lids, semi-air-permeable Parafilm, or stainless steel mesh to allow non-restricted ventilation. Gas concentrations in these arenas and termite behavior and mortality were determined over time. During the study no visible ataxic termite behavior or significant mortality was observed within Parafilm or stainless-steel-mesh capped arenas. In contrast, tightly sealed arenas quickly accumulated significantly more metabolic gases compared with the other arena configurations, causing obvious abnormal termite behavior within 96 hours after these arenas were sealed. Within 8 to 10 days 100% moribund or dead termites occurred in tightly sealed arenas, whereas termites in semi-permeable membrane or non-restricted ventilated arenas were behaving normally. Excess gas accumulation in tightly sealed arenas first elicited negative effects between 96 and 144 hours. Our results show that tightly sealed bioassay arenas must be avoided, even for short-term studies lasting only a few days, as metabolic gases accumulate rapidly, causing negative effects on termites beginning within 4 to 6 days.