The effect of the 21-aminosteroids U74500A and U74389F, alone and in combination with the NMDA receptor antagonist MK-801, on traumatic neuronal injury was quantitatively assessed in murine neocortical cell cultures. Consistent with prior observations, a mechanical insult to the culture monolayer resulted in widespread neuronal death over the following 24 h. Treatment with either U74500A or U74389F provided moderate protection, reducing neuronal death as measured by lactate dehydrogenase release by 25-50%. This effect was most consistent when these agents were preincubated for 2 h prior to injury. Combined treatment with a 21-aminosteroid plus the NMDA receptor antagonist MK-801 reduced injury more than either drug alone. Approximately 40% of the neuronal death occurring in the presence of MK-801 was blocked by concomitant treatment with 10 microM U74500A or U74389F. These results suggest that free radicals may contribute to cell death in this in vitro model of traumatic neuronal injury.