Earlier studies of axonal cytoskeletal responses to stretch injury in the guinea pig optic nerve, a model of nondisruptive axonal injury such as occurs in human diffuse axonal injury, have demonstrated different cytoskeletal responses between the smallest and largest axons. But these form only approximately 3% of the total number of axons in the optic nerve. It was then posited that the pathology described in the latter axons may not be representative of the pathology in the majority of axons after stretch injury. In order to test this hypothesis, we carried out a quantitative, morphological analysis of structural changes in the cytoskeleton of intermediate (axonal diameter of 0.5-2.0 mM) sized axons at 4 h after stretch injury. Neurofilaments in axons up to 1.00 microm in diameter increased in number and in axons up to 1.50 microm diameter were compacted. This did not occur in larger axons (diameter of 1.51-2.00 microm) in the present study. However, there was focal compaction of neurofilaments in some of the larger fibers at sites where the integrity of the axolemma was lost. The response by microtubules to stretch injury differed from that of neurofilaments in that there was an increased spacing between microtubules and a loss of their number in axons of >1.51 microm diameter. We provide quantitative, morphological evidence (a) that the neurofilamentous cytoskeleton of different sized axons responds in different ways to stretch and (b) that the response by microtubules differs from that of neurofilaments.