The specificity of alloantibodies (alloAb) and their clinical significance in association with T - /B + flow cytometry crossmatch (FCXM) in kidney transplantation are not clearly defined. This study was undertaken to examine the HLA specificity and clinical relevance of Ab causing B + FCXM in pre-transplant (final XM) recipients' serum samples. Final FCXM serum samples were analyzed from 457 renal transplant patients followed for 10 months post-transplantation. Two hundred and sixty patients had T - /B + final FCXM. The control group included 197 recipients with T - /B - FCXM at time of transplantation. Class I/class II PRA and specificity of anti-HLA class I and class II Ab in final FCXM serum samples were analyzed by FlowPRA Class I Screening Test and FlowPRA Class II Screening Test. We found no correlation between graft outcome and pre-transplant T - /B - and T - /B + FCXM status. Additionally, we observed no clinical relevance of B + FCXM in retransplant patients. However, MCS ≥ 200 in B + FCXM retransplant recipients was associated with anti-class II Ab to previous mismatches in regrafted patients (n = 46). This finding was confirmed by specificity analysis of anti-DR/DQ Ab in patients with high (≥ 15%) class II PRA. In 63% (12 of 19) of retransplants having T - /B + FCXM, we defined the specificity of alloAb to first graft mismatched class II antigens. In contrast, anti-class II Ab was detected in only 5.7% (2 of 35) of single-graft recipients with different PRA values. Significantly greater MCS (240 ± 61 vs. 163 ± 48; p = 0.022) was observed in retransplant patients having short (≤ 5 m) previous graft survival time (PGST) than in those with long PGST (≥ 5 m). Only 2% of retransplant recipients with B + FCXM had non-HLA Ab. In contrast, the overwhelming majority of primary recipients had no detectable alloAbs. No significant difference in class I PRA was found between B - and B + FCXM recipients. However, class II PRA was significantly higher in patients having B + FCXM (p = 0.028). Collectively, these data show that MCS intensity is not always a reliable criterion for anti-HLA Ab detection because of the presence of non-HLA Ab. These results can be explained by low titers of anti-class II Ab, at which concentration these Ab cannot produce a deleterious effect. FlowPRA and Flow screen beads appeared to be reliable and sensitive methods for detection and specificity analysis of anti-class II alloAb.