Normal human endometrial stromal cells (ESCs) stimulated with 8-Br-cAMP secrete significantly large amounts of PRL and granulocyte colony-stimulating factor (G-CSF), whereas unstimulated stromal cells secreted little. However, there is no relation between PRL and G-CSF levels secreted from the stimulated cells, which suggests that PRL-secreting stromal cells may not completely coincide with the G-CSF-producing stromal cells. Recently we found that macrophage colony-stimulating factor (M-CSF) inhibits 8-Br-cAMP-induced decidualization, differentiation to prolactin (PRL)-secreting cells, by suppressing viable decidualizing cells. Therefore, we investigated the effects of M-CSF on G-CSF secretion from normal human endometrial stromal cells using an in vitro decidualization activity assay. M-CSF did not show any significant effects on viable cell numbers of unstimulated ESCs, while M-CSF dose-dependently enhanced G-CSF release from the non-decidualized stromal cells. A high concentration of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells co-stimulated with 8-Br-cAMP and M-CSF, although G-CSF release from the co-stimulated stromal cells was not affected by M-CSF. Moreover, M-CSF did not affect viable cell numbers, PRL secretion or G-CSF secretion of 8-Br-cAMP-stimulated cells. These results indicate that M-CSF enhances G-CSF secretion from 8-Br-cAMP-unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells, thus suggesting that there exists a functional subpopulation of G-CSF-secreting stromal cells that are different from the predecidualized ESCs that differentiate into PRL-secreting ESCs under stimuli with 8-Br-cAMP. Hence, M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells in an autocrine or a paracrine manner.