Limb regeneration in the short-toes axolotl is impaired. Our goal was to characterize the regeneration process in this mutant by histological and immunocytochemical methods. Previous research indicates that age and a defective basement membrane may be instrumental factors in short-toes axolotl regeneration (Del Rio-Tsonis et al. [1992] Proc. Natl. Acad. Sci. U.S.A., 89:5502-5506). The present results show that limb regeneration can occur even in older (1-2-year-old) short-toes axolotls. The process was always significantly delayed, but the time required for complete regeneration varied. Even so, the basement membrane of short-toes regenerates showed no differences in thickness or shape compared with wild-type regenerates. Distally amputated short-toes limbs gave rise to more digits in the regenerate, indicating that regeneration may be somewhat dependent on the level of amputation. Since extracellular matrix (ECM) remodeling occurs extensively during regeneration, we compared the ECM of the short-toes and wild-type regenerates using monoclonal antibodies (mAbs) MT2 and ST1 (Tassava et al. [1996] Wound Rep. Reg., 4:75-81). The short-toes regenerates showed decreased reactivity to mAb MT2, which identifies type XII collagen, an ECM protein that is normally unregulated during regeneration, and increased reactivity to mAb ST1, which identifies a limb ECM component that typically undergoes breakdown in the distal stump. Thus, impaired regeneration in the short-toes axolotl is correlated with impaired ECM remodeling in the distal limb stump. This supports the view that ECM remodeling plays an important role in regeneration.