Improved tolerance of splenectomized patients with Hodgkin's disease (HD) to radiotherapy and chemotherapy has been reported. The present study was undertaken to determine the effects of splenectomy and nitrogen mustard (NM) on colony-forming cells (CFC's) of bone marrow cells obtained from CF1 male mice by in bitro agar-gel technique. Splenectomized mice were given NM intraperitoneally on day 11. On day 15, they were sacrificed and the bone marrow was cultured with a source of colony-stimulating factor (CSF). Spleen extract was prepared by grinding spleens from CF1 mice. On the eighth day of incubation, significantly higher numbers of CFC's were found in splenectomized animals at 1% confidence level (F Test) compared with the nonsplenectomized animals. Both splenectomized and non-splenectomized mice had a greater colony response after NM (at 5% confidence level) than saline-treated controls. Maximum numbers of colonies were obtained in the nustard-treated asplenic animals. Splenic extract, as well as extracts from other organs, when added to the culture plates resulted in inhibition of colony formation. The significance of in vitro inhibition after addition of organ extract is uncertain.