Two distinct cell morphologies were appreciated and separated in a long-established culture line (CHP-100) of human neuroblastoma. Both cell types carried chromosomal markers characteristic of neuroblastoma cells and the parent line; in addition, separate karyotypic changes in each cell type established them as separate and enriched populations. A small, refractile cell, designated CHP-100-S, was present and formed numerous cytoplasmic processes. A distinctly larger cell, CHP-100-L, was less refractile and lacked processes. The two cell types exhibited marked differences in adhesive properties in vitro. CHP-100-L adhered tightly to the culture flask and required enzymatic treatment for removal; CHP-100-S adhered loosely and could be harvested into the medium by simply tapping the flask. These two harvesting procedures were used to obtain highly enriched populations of each cell type, both of which proved to be tumorigenic in the nude mouse. In vitro, no significant difference in growth rates was observed between CHP-100-S (doubling time, 26 hr) and CHP-100-L (21 hr). However, in the nude mice, following inoculation of equal cell numbers, CHP-100-L cells grew much larger tumors than did CHP-100-S cells (3- to 100-fold increases over 25 days). Local invasion was also noted more frequently with the CHP-100-L explants. Reculturing of the mouse explants showed that the distinct cell morphologies were maintained even after multiple passages. The presence of heterogeneous cell populations in single tumors is of much potential importance for the clinical and biological behavior of neoplasms. The present data establish cultured human neuroblastomas as one model for studies of cell heterogeneity and suggest potentially important ramifications for the different cell types observed in the growth patterns of this neoplasm.