SummaryHuman multinegative (CD3 −4 −8 − 19−; MN) thymocytes proliferate optimally in response to anti‐CD2 plus anti‐CD28 mAb plus PMA or IL‐7. The role of CD45 was assessed by the addition of mAb to a CD45 common determinant, or to CD45RA. MN thymocytes are unresponsive to anti‐CD2 mAb. Co‐stimulation with anti‐CD45RA generated 1.6–5.7‐fold enhancement of a proliferative response, with maximal enhancement by cross‐linkage of CD45RA molecules. The response to anti‐CD2/28 mAb was reproducibly enhanced only by immobilized anti‐CD45RA. Cross‐linking of CD45RA and CD28 through the use of heteroconjugates of mAb did not enhance the co‐stimulation by CD45RA. The most marked enhancement by anti‐CD45RA occurred in suboptimal activation conditions. In contrast, the response to anti‐CD2 or anti‐CD2/28 was inhibited by mAb to CD45 common determinants (anti‐CD45) in the presence or absence of PMA or IL‐7, with the most profound inhibition (6–8‐fold) detected in optimal proliferative conditions. Cross‐linking of CD45 and CD28 through heteroconjugates of mAb was required as soluble anti‐CD45 or immobilized anti‐CD45 were unable to mediate inhibition. This inhibitory effect of (anti‐CD45 × 28) was specific to MN thymocytes as no inhibition was detectable when peripheral blood T cells were treated with anti‐CD2/28 and the same heteroconjugate. The differential effects of anti‐CD45 and anti‐CD45RA may reflect either CD45 heterogeneity on MN thymocytes, or the physical modulation of a single CD45 molecules by interactions at different epitopes, and the avidity of the relevant CD45 mAb for thymocyte CD45 isoforms may play a role. We conclude that the molecular associations between signal transduction molecules on progenitor thymocytes differ significantly from those assembled on peripheral blood T cells.