Cell culture methods can allow investigation of the mechanisms responsible for immunotoxicity. Unfortunately, natural killer (NK) cells in rodent splenic cultures rapidly lose their cytolytic function. It is not known if death of NK cells or loss of function in viable NK cells is primarily responsible for this loss. Flow cytometry and an assay of NK cell lytic function were used to address this issue and to determine if NK cell viability could be maintained by adding selected cytokines or a caspase inhibitor to the cultures. Total cells and NK cells in untreated 18 h cultures were 79 +/- 1% and 25 +/- 2% viable, respectively, and these cultured splenocytes caused only 4 +/- 1% specific release of (51)Cr from YAC-1 target cells. Cultures including polyinosinic:polycytidylic acid (poly I:C) or IL-2 had increased NK cell viability (43 +/- 2%, 47 +/- 1%) and function (58 +/- 2 and 43 +/- 1% specific release). IL-15 significantly increased NK cell viability, but not function. Previous studies demonstrated that treatment of mice with immunotoxicants such as ethanol or corticosterone diminishes NK cell activation in vitro in response to poly I:C. To determine if alterations in viability are responsible for this decreased NK cell activity, lytic function and NK activity were measured in cultures of splenocytes treated in vivo or in vitro with ethanol and/or corticosterone. Some treatments reduced IL-2 or poly I:C-enhanced lytic activity in vitro, but there was no clear relationship between these changes in function and changes in the percentage of viable NK cells. Thus, immunotoxicants that suppress NK cell activation can be investigated in vitro because commonly used activating stimuli also permit NK cell survival. However, no agents were identified that could maintain NK cell viability and function in culture (without activation) to allow investigation of the direct effects of immunotoxicants on basal NK activity in vitro.