A methodology for rapid isolation of neuroblastoma cells from marrow with metastatic neuroblastoma cells was developed using a cocktail of five antibodies and magnetic microspheres coated with secondary antibodies. Cells bound to microspheres were released by brief exposure to chymopapain, followed by repeated culture of released cells in serum-supplemented Dulbecco's modified Eagle's medium and selection for adherent cells. Using this methodology, over 35 primary cell lines were obtained free of contaminating normal cells. Detailed analyses of over 14 cell lines revealed gross differences in cell phenotype, size, morphology development of neurite processes, and doubling time (40 to 80 h). All cell lines expressed the M(r) 145,000 neurofilament, and a few expressed the M(r) 200,000 neurofilament, with very little or no expression of the M(r) 68,000 neurofilament. Eight % of all cells lines had near-diploid DNA content. High expression of the MDR-1 protein was detected in six of the 22 cell lines tested. Great heterogeneity was observed in the expression of N-myc oncoprotein, with ten of 13 patients overexpressing the protein. c-myc oncoprotein was also expressed in all cell lines; however, the level of expression was 4- to 10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc, whereas N-myc was localized to the nucleus and to the cytoplasm.