Previous studies in the mouse have strongly implicated colony-stimulating factor-1 (CSF-1) in the regulation of placental development. In this study of human pregnancy, we detected CSF-1 in serum, endometrium, placenta, chorion, amnion, and amniotic fluid, with significant increases in serum and endometrial samples from the first trimester compared to levels in nonpregnant controls. CSF-1 mRNA was demonstrated in all of these tissues, except amnion, with a significant increase within the first trimester endometrial samples over nonpregnant control values. In addition to the major 4.0-kilobase mRNA, other species of CSF-1 mRNA were detected, which were shown to be due to alternative splicing within exon 6 and the alternative use of exon 9 or 10. In the endometrium, CSF-1 was localized to glandular epithelial and endothelial cells. In first trimester placenta, CSF-1 was in the cytotrophoblasts lining the villous core and in the cytotrophoblastic shell. During the second trimester, CSF-1 was localized to villous mesenchymal cells. By the third trimester, CSF-1 was only detected in cells lining the villous vessels. The detection of CSF-1 during gestation strongly supports a role for CSF-1 in the regulation of placental function in humans by autocrine and/or paracrine mechanisms.