Fibroblasts and neuroblastoma cells kept in monolayer cultures, as well as surface spreads of mitotic chromosomes, were stained with picrosirius red. Red staining (in normal light) and optical anisotropy of the stained structures (in polarized light) were observed intracellularly and in the chromosomes. The intracellular and extranuclear birefringence induced by staining with sirius red could not be abolished by digestion with collagenase prior to staining, or by treatments used to disrupt microtubules (vinblastine, colcemid) or microfilaments (cytochalasin B). We therefore propose that the parallelly-arranged intermediate filaments are responsible for the optical anisotropy induced by sirius red staining in these cells. In addition, the spatially oriented scaffold of chromosomes can be detected by sirius red-induced birefringence. These data argue against the collagen-specificity of picrosirius red staining and of the birefringence induced by this technique. Our results also suggest that picrosirius red staining combined with polarized light microscopy can be used to study the spatial orientation pattern of the intermediate filaments and chromosome scaffold.