Few prospective studies for Lyme borreliosis (LB) have been conducted among subjects at risk to acquire this disease during their work when wearing well-defined protective clothing against tick-bites. We prospectively compared the rate of seroconversion and the occurrence of clinical manifestations of LB among 2 groups of Dutch military. The at risk-group worked predominantly outdoors, and the control-group conducted predominantly indoor activities. During outdoor work, the at risk-group wore long sleeves, long trousers, closed around the ankles and high boots. Initial serum samples from 2158 military were collected immediately after entering the service. From 932 military (at risk-group: 469, control-group: 463) a second sample was drawn 9 to 12 months later. Sera were tested for IgG and IgM antibodies by ELISA. Four persons (0.9%) in the at risk-group and 10 persons (2.2%) in the control-group seroconverted for IgG and/or IgM antibodies against B. burgdorferi. This difference was not statistically significant. Only 1 person who had seroconverted had noticed an erythema migrans. In conclusion, military performing outdoor activities have a low risk for infection with B. burgdorferi, most likely because they wear protective clothing. Among seroconverted persons clinical manifestations of Lyme borreliosis occurred rarely.