Pertussis toxin (PT) inhibits invasiveness of T-cell hybridomas in vitro and metastasis formation in vivo. We present evidence for the hypothesis that PT interferes with functional activation of LFA-1. Invasion by TAM2D2 T-cell hybridoma cells of fibroblast monolayers was completely blocked by PT pretreatment, but the cells regained invasiveness in the presence of Mn2+, which activates LFA-1. This invasion was blocked by anti-LFA-1 mAb, and Mn2+ did not stimulate invasiveness of LFA-1-deficient TAM2D2 mutants. TAM2D2 cells did not adhere to surfaces coated with the LFA-1 counterstructure ICAM-1, but Mn2+ induced adhesion. Hence, LFA-1 on TAM2D2 cells requires activation before it can participate in the invasion process. The hypothesis is further supported by the slightly different results obtained with the TAM8C4 T-cell hybridoma. PT inhibited invasion strongly but not completely. This reduced invasion was increased by Mn2+. TAM8C4 cells did adhere to ICAM-1, but Mn2+ enhanced adhesion. Thus, part of LFA-1 on TAM8C4 cells is constitutively active, allowing for some PT-insensitive invasion, but further activation is required for optimal adhesion and invasion. PT blocks G-protein-mediated signals, suggesting that an extracellular factor is involved. This is not a serum component or an autocrine motility factor, since the PT effect was serum-independent, and PT did not inhibit motility. Therefore, it is probably produced by the fibroblasts, and either secreted or associated with the cell surface. These results are in line with the hypothesis that a fibroblast constituent activates LFA-1 via a PT-sensitive G-protein and thus stimulates invasion of T-cell hybridomas into the fibroblast monolayer.