With the development of transgenic and specialized mouse strains, there is an increased need for inhalation exposure systems designed for smaller exposure groups. An inhalation exposure chamber, designed specifically for the exposure of up to 40 mice, was characterized. The chamber was fabricated from 0.32-cm-thick ((1)/(8)-in) aluminum sheets with outside dimensions of 61 cm long by 32 cm high by 34 cm deep, resulting in an internal volume of 65 L. Two stainless-steel open-mesh cages, separated by an absorbent barrier, can be stacked within the central portion of the chamber. Access is provided through a gasketed door with a safety-glass face. Tests were performed to determine the chamber leakage rate, degree of mixing, and spatial variation of two aerosols within the chamber. Results indicated that the fractional leakage rate was 0.0003 min(-1), well below a reported criterion for an operating chamber. Chamber operation gave similar mixing performance with, or without, use of an interior fan. For aerosols with a mass median aerodynamic diameter (MMAD) of 2.56 micro m and 3.14 micro m, the spatial variation of particulate matter concentration resulted in coefficients of variation (CVs) of 4.8% and 11.0%, respectively. These CV values are comparable to those obtained from similar studies involving other inhalation exposure chambers.