The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin‐12 (rhIL‐12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL‐12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B‐blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL‐12 treated patients, increased IFN‐γ mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL‐12 treatment. In conclusion, these data indicate that after i.t. IL‐12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients. © 2008 Wiley‐Liss, Inc.