The mixed lymphocyte culture (MLC) is known to react to major histocompatibility complex disparities but is insensitive to minor histocompatibility antigens. To determine which modifications could amplify reactivity to these antigens, we have studied the effects of presensitization on normally nonresponsive MLC in dogs. Lymphocytes, primed in vitro to and restimulated with cells of a DLA-identical sib, did not react to these specific cells, in contrast to normal proliferation to other stimulators. However, lymphocytes obtained after in vivo sensitization against a DLA-identical donor showed increased reactivity to the donor in approximately half of the recipients. In comparison to primary MLC responses between DLA-mismatched individuals, the [3H] thymidine uptake was less important in this reaction, and peak incorporation was usually not accelerated. This response appeared to be specific in that responsiveness to third-party cells was not increased, and, alternatively, immunization against a DLA haplo-identical donor did not modify reactivity to DLA-identical cells. This increased reactivity after alloimmunization may reflect differences in minor histocompatibility antigens although the skin graft survival time was not significantly shorter in MLC responders than in nonresponder recipients. It seems that in vivo priming is required to expand the number of cells specific for these antigens and to enable detection of proliferation in MLC.