Positive stress has been described in the literature but not well characterized experimentally. This experiment was designed to test the hypothesis that environmental enrichment and housing density in C57BL/6 female mice modulate immune responses to acute exercise stress. A 2 x 2 x 2 factorial design was used where enrichment (enrich), nonenriched (nonenrich), group housed (group), individually housed (ind), and stress (stress) or no stress (nonstress) acted as the independent variables. Enrichment involved a 7-week exposure to in-cage running wheels and a variety of cage objects. Ninety minutes after treadmill exercise stress at 25 m/min, 4 degrees slope, for 30 min (or no exercise stress) mice were sacrificed, and splenocyte blastogenesis to the T-cell mitogen concanavalin A (Con A), splenic T-cell subset enumeration, and percent live/dead splenic cells by flow cytometry were evaluated. Results showed significant interaction effects for mitogen responses, percent Ly2+ subset, and percent live/dead splenocyte responses. Proliferation to Con A was higher in nonenrich group-housed animals than other groups. Percent live/dead cell analysis revealed a significant housing x stress interaction with fewer percent live and higher percent metabolically stressed splenocytes obtained from ind-stressed mice than other groups. These data suggest that enrichment and housing density are important factors influencing immune responses in the basal state, and in response to exercise stress.