Serotonin has been shown to be involved in the production of bystander signals by irradiated cells. In this study we examined the levels of serotonin in 10 different batches of commercially available fetal calf serum and correlated the serotonin levels with the toxicity of medium harvested from irradiated cells (ICCM) using a standard medium transfer colony-forming assay. The serotonin levels in the serum varied widely between batches, and the levels correlated directly with the toxicity of the harvested ICCM. Three serum samples had levels of serotonin below 25 ng/ml, and these did not show medium transfer bystander effects. Exposure of serum samples to normal daylight reduced serotonin levels significantly. We suggest that serum batch variability may underlie much of the observed interlaboratory variation in the ability to produce bystander effects and further suggest that serum batches should be protected from light and prescreened for their ability to produce a bystander effect using a positive control cell line.