Trypanosoma musculi cultivated in medium containing serum alone or in the absence of fibroblasts in vitro were transformed into rounded, immotile cells, incapable of division and infectivity. Only in close contact with fibroblasts could the parasites survive and grow indefinitely. This report established the identity of the splenic 'sustentacular' cells as fibroblasts and utilized immunocytochemistry to demonstrate the putative cytoskeletal and membrane-associated molecules that may be involved in the control of growth and division, and apoptosis of T. musculi in vitro. The results indicated that cells that reacted intensely for fibroblast growth factor (FGF) also displayed a complex cytoskeletal system of F-actin bands underlying the plasma membrane of the fibroblast cell body and its numerous processes. Among the cytoskeletal and membrane glycoproteins, fibronectin, I-CAM, laminin, occludin, vinculin and desmin were most prominent. Fibronectin was most highly enhanced on the cell membrane and deposited as 'finger prints or tracks' on the extracellular culture surfaces. Transmission and scanning electron microscopy confirmed the intimate contact between trypanosomes and fibroblasts, however, neither membrane fusion or junctions were apparent. Our results suggested that a fibroblast-derived, membrane-associated factor appeared to be the putative growth regulator and apoptosis inhibitor in co-cultures of spleen-derived fibroblasts and T. musculi.