Human and nonhuman primates have multiple DR B1 and non-B1 alleles. However, the role of mismatched DR non-B1 alleles in primary alloimmune responses is not well understood. Macaques, which share close DNA homologies with human MHC genes and have a high number of β-chain genes in the DR subregion, are preeminent preclinical models for immunologic studies of transplant tolerance and immunosuppression. In this study, we examined the effect of allogeneic MHC Class II DRB mismatches in Th1- and Th2-like cytosine responses elicited in one-way MLR cultures in rhesus macaques. An ELISPOT method was used to estimate cytokine secretion at the single cell level. Molecular typing for DRB1 and DR non-B1 alleles was performed by a moderate-high resolution PCR-SSP method using a panel of 55 primer pairs covering 74 DRB alleles and clusters. Of 35 unrelated combinations, 66% had multiple (≤2) allelic MM at DRB1 and DR non-B1 with no significant correlation between numbers of DRB1 and DR non-B1 mismatches. Pairs with 1 or 0 MM were assigned to a mono/null MM group to obtain sufficient numbers for statistical analysis. The pairs differing by multiple vs. mono/null DRB1 MM showed no significant difference in cytokine prevalence (P = 0.69). In contrast, high IFN-γ/IL4 SFC ratios were noted in pairs with multiple vs. mono/null DR non-B1 MM (p = 0.0009). IFN-γ/IL-10 spot forming cell (SFC) ratios were consistent with IFN-γ/IL-4 SFC ratios (r = 0.98). Multiple DR non-B1 mismatches showed a trend towards higher MLR proliferative responses, although showed a trend towards higher MLR proliferative responses, although the stimulation index did not reflect the dominant cytokine response. These observations suggest a bias towards Th1-like cytokine production under allostimulation with multiple DR non-B1 gene products. Further study of the primary structure of DR non-B1 determinants may be helpful in understanding the fine molecular mechanisms governing the regulation of cytokine profiles during allostimulation in primates.