Interactions among neuronal subpopulations determine brain development and function. The present study illustrates the ability to noninvasively and selectively lesion targeted subpopulations of neurons in a highly specific, temporally defined, and geographically localized manner. This method provides a fundamental advance toward rigorous investigation of the importance of identified neuronal subtypes. Projecting pyramidal neurons of rats and mice were targeted by retrograde transport of latex nanospheres from contralateral motor cortex containing a nontoxic chromophore (chlorin e6) that produces cytotoxic singlet oxygen during photoactivation by deeply penetrating 670-nm light. Geographically defined regions of cortex were exposed to laser illumination at 670 nm to activate singlet oxygen production by the intracellular chromophore, and animals were sacrificed from 4 h to 7 days following laser exposure. Brains were processed to display degenerating neurons using sensitive silver-staining procedures. Selective damage occurred to pyramidal neurons with known callosal projections, solely within layers II/III and V of the illuminated region. Such subpopulation specificity provides models for neural transplantation and analysis of anatomically distributed neuronal networks.