Three cell lines from resident macrophages of BALB/c mice and four from activated macrophages of the same strain were isolated by infection with simian virus 40 (SV40). A majority of these cells showed dependency on L cell-conditioned medium (LCM), which is necessary for proliferation of normal macrophages in vitro. Somatic cell hybridization was applied in the study of macrophage growth responsiveness. A macrophage cell line (BR15) with strict dependency on LCM for growth was fused to a Chinese hamster cell line (hs222-16); it was found that dependency on LCM was a dominant trait in the hybrids. Following fusion of a macrophage cell line (BAM3) which grew without LCM to hs222-16, a large number of colonies appeared in the selection medium containing LCM. Four hybrids not requiring LCM for growth were selected in an LCM-free culture, and their hybrid properties were examined. Three out of the four hybrids secreted colony-stimulating factor (CSF) constitutively, whereas the fourth secreted no CSF. The level of acid phosphatase activity in the hybrids was higher than in the parent cells. Two peaks of CSF activity were observed after gel filtration chromatography of conditioned medium: One was eluted at molecular weight of 36,000 and the other at 17,000.