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In preparation for functional analyses, a study of the binding of H-2Kb-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2Kbm mutant cells and on FOR-treated H-2Kb cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KPb-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2b cells as indicated by cold target inhibition studies. The H-2Kb-specific CTL were probably reactive with “conformational” determinants of H-2Kb, which are dependent on the integrity of both the a1 and the a2 domains of the H-2Kb molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the a1 domain without affecting conformational-type CTL determinants.