The humoral immunoreactivity in Lyme borreliosis is a well characterized parameter for establishing the diagnosis of a Borrelia burgdorferi (Bb) infection. Since patients with seronegative Lyme borreliosis have been described, lymphocyte proliferation tests may be used for detecting patients who only develop a cellular immunoreactivity against Bb organisms. We performed a the lymphocyte proliferation assays in order to determine the cellular immunoreactivity to these spirochetes in given patients with histologically confirmed diagnosis of erythema migrans (EM), acrodermatitis chronica atrophicans (ACA), and for control purposes patients with Lyme disease non associated dermatoses (NLDH) and healthy volunteers (G). Stimulation index was defined as 3H thymidine-uptake in peripheral mononuclear cells, calculated as quotient dpm (stimulated cells)/dpm (unstimulated cells) (dpm = disintegrations per minute). Stimulation indices > 10 were considered positive. We detected elevated cellular immunoreactivity of peripheral mononuclear cells in 33% EM-patients (3/9) and in 23% (3/13) ACA patients tested. Patients tested before and after antibiotic therapy showed a significant decrease of stimulation index after antimicrobial treatment (p < 0.05). Patients the EM, who are mostly seronegative at the onset of cutaneous eruption exhibit in a third of patients an elevated cellular immune response to Bb. Therefore lymphocyte proliferation assays can be recommended as an additional test system in case of lack of serological response. The significant decrease of stimulation index after antimicrobial therapy indicates for downregulation of the cellular immune response to these spirochetes investigated, and counts for the specificity of this test system.