Using an in vitro monolayer invasion assay (MIA), we analyzed the interaction of human myeloid cell lines representing different maturation stages with murine fibroblastic monolayers. After 24 h of coculture, only promonocytic U937 cells invaded the monolayer to an appreciable extent in contrast to less-differentiated KG1a, KG1, and HL-60 cells and more mature THP-1 cells. Human interferon-gamma (HuIFN-gamma) treatment was found to induce maturation in the U937 line and resulted in a considerable reduction of interaction with the monolayer. Thus, the capacity of myeloid cells to interact with a fibroblastic monolayer is restricted to a specific maturation stage. Interaction of U937 cells was also abolished when they were treated with pertussis toxin (PT), an agent known to induce monocytosis in vivo, indicating that the MIA may serve as an in vitro simulation of the extravasation of blood borne cells. Finally, although both HuIFN-gamma and PT are able to block cell spreading in the MIA, no effect could be seen on the capacity of U937 cells to phagocytose.