We examined cytochrome oxidase (CO) activity in striate cortex of four macaque monkeys after monocular enucleation at ages 1, 1, 5, and 12 weeks. These animal experiments were performed to guide our interpretation of CO patterns in occipital lobe specimens obtained from two children who died several years after monocular enucleation during infancy for tumor. In the macaques, the ocular dominance columns were labelled by injecting [3H]proline into the remaining eye. After enucleation at age 1 week, ocular dominance columns were eliminated in layer IVcβ, resulting in a uniform pattern of autoradiographic label and CO staining. However, columns could still be seen in wet, unstained sections and with the Liesegang silver stain. Autoradiographs through layers IVcα and IVa showed residual, shrunken columns belonging to the missing eye, indicating that enucleation has less drastic effects in these layers. In the two human cases, enucleation at age 1 week also resulted in uniform CO staining in layer IVc. In the macaque after enucleation at age 5 weeks, ocular dominance columns belonging to the missing eye were severely narrowed, but still occupied 20% of layer IVcβ. CO revealed wide, dark columns alternating with thin, pale columns in layer IVcβ. The CO pattern and the columns labelled by autoradiography matched perfectly. After enucleation at age 12 weeks, only mild shrinkage of ocular dominance columns occurred. Enucleation at ages 1, 5, and 12 weeks did not alter the pattern of thin-pale–thick-pale stripes in V2. The main findings from this study were that (1) CO histochemistry accurately labels the boundaries of columns in layer IVcβ of macaque striate cortex after early monocular enucleation, making it a suitable technique for defining the critical period for plasticity of ocular dominance columns in human striate cortex; (2) enucleation causes more severe shrinkage of ocular dominance columns than eyelid suture; (3) early monocular enucleation obliterates ocular dominance columns in layer IVcβ, but their pattern remains visible in wet sections and with the Liesegang stain; and (4) enucleation does not affect CO staining in V2.