Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from the deficient activity of acid sphingomyelinase (ASM). Type A NPD is characterized by the absence of residual ASM activity, massive accumulation of sphingomyelin and cholesterol within lysosomes, and a rapid, neurodegenerative course that leads to death by 3 years of age. In contrast, type B NPD patients have low, but detectable, levels of residual ASM activity and little or no neurologic disease. Thus, individuals with type B NPD may survive into late adolescence or adulthood and are considered excellent candidates for somatic cell gene therapy. To facilitate the development of gene therapy for this disorder, a novel procedure was devised to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Type B NPD cells were transduced with retroviral vectors expressing ASM, labeled with lissamine rhodamine sphingomyelin (LR-SPM), and subjected to preparative fluorescence-activated cell sorting (FACS). Two non-overlapping cell populations were isolated, corresponding to enzymatically corrected (i.e., low fluorescence) and noncorrected (i.e., high fluorescence) cells. Quantitative PCR analysis demonstrated that the enzymatically corrected cells were enriched for vector sequences. Moreover, the corrected cells could be regrown and continued to express high levels of ASM activity after numerous passages, consistent with the fact that they were stably transduced. Notably, coculture of FACS-sorted, overexpressing cells with untreated type B NPD fibroblasts resulted in a homogeneous cell population with low fluorescence whose FACS distribution overlapped that of the corrected cells. Computerized fluorescence microscopy confirmed that nearly all of these cocultured cells expressed ASM activity and could hydrolyze LR-SPM. Thus, this procedure provides a rapid, preparative method to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Because transduced cells were able to cross-correct untreated cells, these results also suggested that “bystander correction” may occur following autologous transplantation of retrovirally transduced cells into NPD patients, providing further rationale for the development of gene therapy for this disorder.