The aim of this investigation was to explore whether the occurrence and the magnitude of radiation-induced, medium-mediated bystander effects could be influenced by the time of transfer of secreted bystander factors. HaCaT cells were exposed to 0.1 and 1.0 Gy of gamma radiation. These doses did not induce a significant reduction in the clonogenic survival of irradiated cells compared to controls. Bystander cells either were co-cultured with irradiated cells or received medium from irradiated cells. The bystander effects analyzed included end points related to survival (clonogenic potential and cell proliferation) and DNA damage (micronucleus induction and gamma-H2AX formation). The bystander effects we investigated either were lacking or varied from potentially protective to detrimental responses in relation to the dose of radiation and the time between irradiation of donor cells and bystander exposure. Our results suggest that the experimental time schedule is important for both the occurrence and the detection of bystander effects in vitro.