Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The aim of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when exposure occurred via the drinking water. Consistent with human exposure, female B6C3F1 mice were exposed to chloroform-containing drinking water at 2.5, 10, 25, 100, and 250 ppm for 28 days. The examined endpoints included the effects of chloroform on body and organ weights, water consumption, hematology, innate immunity, humoral immunity, and cell-mediated immunity. The functions of natural killer, B-, and T-cells were not altered by chloroform in drinking water at the concentrations tested, except that an increase in splenocyte basal proliferation was observed at chloroform levels of 100 and 250 ppm. Following chloroform administration, there was a decreased number of circulating neutrophils in the blood in all treatment groups, but neutrophil function in lung homogenates, as evaluated using an assay for myeloperoxidase activity following lipopolysaccharide and N-Formyl-Met-Leu-Phe stimulation, was not compromised. Further, the results of host resistance to Listeria monocytogenes infection also suggested that neutrophil function was normal. At the highest treatment level of chloroform (250 ppm), erythrocyte number and hemoglobin levels were significantly decreased. Some significant changes were also observed for body weights, water consumption, and organ weights; however, most of these effects were only observed at the highest treatment level of chloroform (250 ppm). Taken together, the results demonstrate that while chloroform administered via the drinking water affects body weight and selected hematological parameters at high dose levels, overall immune responses, as measured in several tests for immune function, are not compromised.