A highly heterogeneous cell line, IIB-MEL-J, was established from a human metastatic melanoma. This cell line contains small cells, dendritic cells, and megacells with multiple nuclei. IIB-MEL-J expresses S 100, cytokeratin intermediate filaments and the gangliosides GD2 and GD3. It requires growth factors (insulin, EGF, and transferrin) and antioxidants for optimal growth. When plated under optimal conditions, IIB-MEL-J grows with a doubling time of 70-80 hours. The cells may be fractionated by Percoll gradient centrifugation into several subpopulations (A, B, and C) with different characteristics. Subpopulation A is the slowest growing, and most of the DNA-synthesizing cells are concentrated in fractions B and C. Every subpopulation expresses S 100 and cytokeratin intermediate filaments, whereas only subpopulation B and C express GD2 and GD3. Pigmented cells are concentrated mainly in subpopulation C. Cytogenetic analysis of IIB-MEL-J revealed extensive chromosomal alterations, including a highly heterogeneous chromosome number and chromosomal rearrangements, gains, losses, isochromosomes, and double minutes. This highly heterogeneous cell line may be helpful to study cellular differentiation and interaction between different subpopulations in human melanoma.