Axonal regrowth after injury is accompanied by changes in the expression of tubulin, but the contributions of substrate molecules and neurotrophic factors in regulating these changes in vivo are not known. Adult rat retinal ganglion cells (RGCs) were examined after intraorbital axotomy, after application of a peripheral nerve (PN) graft to stimulate regeneration, and after axotomy and treatment with brain-derived neurotrophic factor (BDNF). After these treatments we used in situ hybridization to study mRNA levels for betaI, betaII, betaIII, betaIVa, and Talpha1 tubulin isotypes. Levels of mRNA for all isotypes were downregulated after intraorbital axotomy. During regrowth of injured RGC axons, mRNA levels for betaII, betaIII, and Talpha1 isotypes were upregulated specifically and dramatically, suggesting that elevated expression of these isotypes is correlated specifically with axonal regrowth. A corresponding increase in betaIII protein levels was detected by immunocytochemistry. The betaI and betaIVa mRNAs were not increased during regeneration. BDNF did not elicit a specific increase in the mRNA levels for the betaIII and Talpha1 isotypes and had only a small effect on mRNA levels for the betaII isotype. Therefore, despite the ability of BDNF to support the survival of injured RGCs and to enhance neurite outgrowth of retinal neurons in vitro, the in vivo application of BDNF alone is unable to induce the program of changes in growth-associated tubulins that accompany regeneration of RGC axons into PN grafts. We speculate that, in addition to BDNF, cooperative signaling with substrate molecules is required to allow RGCs to regenerate and exhibit tubulin isotype switching.