The inbred laboratory mouse may offer the best opportunity to investigate the sensitivity of the mixed lymphocyte reaction (MLR). Although previous investigators have reported transformation of mouse cells, they have not delineated the entire time sequence of the reaction. We have separated mouse lymphocytes from spleen by centrifugation and have successfully incubated them in 20% mule serum for up to 9 days. The one-way and the two-way MLR exhibited peak activity at 5 to 6 days. The response to phytohemagglutinin was maximal on the 3rd to 4th day. Histocompatibility differences at the H-2 locus were detected by both the one-way and the two-way MLR, whereas differences at non-H-2 loci were consistently detected by only the two-way MLR. There was significantly increased activity in the parent vs F1 one-way irradiated MLR, but not in the F1 vs parent irradiated.