OBJECTIVE The reflectance of the retinal nerve fiber layer (RNFL) arises from cylindrical light scattering elements distributed throughout its thickness. To determine whether these elements include axonal microtubules, eyecup preparations of the toad Bufo marinus were treated with the microtubule depolymerizing agent colchicine. METHODS Some eyecups were incubated for various times in different colchicine concentrations and then fixed and prepared for electron microscopy (EM). In other eyecups the reflectance of the RNFL at 440 nm was measured by means of imaging microreflectometry. After a period of baseline measurements, the bathing solution was changed either to one of identical composition (control experiments) or to one containing 10 mM colchicine. Measurements then continued for 2 to 3 hours. RESULTS Quantitative EM showed that colchicine caused the density of axonal microtubules to decrease by approximately one third in 2 hours. Reflectometry showed, in control experiments, that the RNFL reflectance remained near baseline for the duration of an experiment. In contrast, after changing to colchicine solution, the RNFL reflectance declined to a level 37% to 55% below baseline. CONCLUSIONS Microtubules made a major contribution to the 440-nm reflectance of the unmyelinated axons of the toad RNFL and could make a similar contribution to the reflectance of human RNFL.