During rush venom immunotherapy (VIT), about 65% of patients develop large local reactions (LLR) at the application site that last for at least 24 h. However, LLR subside during long-term treatment. To learn more about the provenance of infiltrating cells in late, local skin reactions during VIT, we analyzed the skin infiltrates of 23 Hymenoptera venom (HV)-allergic patients. Punch biopsies were obtained 24 h after s.c. injection of HV allergens from 23 HV-allergic patients and five nonallergic controls. Seven patients did not show LLR at the beginning of VIT. Ten patients had LLR when the dose of HV allergens was increased. Six patients showed reduced LLR after long-term treatment. Immunoenzymatic labeling of the cryostal sections with a panel of monoclonal antibodies was performed by the APAAP method. S.c. application of HV allergens induced a perivascular and periadnexial cutaneous mononuclear cell infiltrate consisting mainly of CD4+, CD45RO+; and HLA-DR+ cells in patients without clinically apparent LLR. In contrast, LLR were associated with a significant increase in total cells, CD4+ cells, CD8+ cells, CD11c+ cells, EG2+ cells, NP57+ cells, HLA-DR+ cells, CD45RO+ cells, CD45RA+ cells, CD23+ cells and CD25+ cells (P < 0.001). Decreased LLR after long-term VIT was correlated with a significantly reduced recruitment of CD4+ cells, EG2+ cells, and CD23+ cells as compared to LLR in the course of dose increases (P < 0.05), whereas the number of CD8+ cells, CD11c+ cells, NP57+ cells, and CD25+ cells remained high. Our data suggest that s.c. injections of HV allergens attract CD4+ helper T cells, of both the naive (CD45RA+) and memory (CD45RO+) phenotypes, to the allergen application site. LLR represent delayed allergic rather than toxic reactions to HV components and might be relevant to the development of clinical protection during VIT.