We report here the detailed cellular and genetic analysis of two new human melanoma cell lines established from the same tumor biopsy. Although derived from the same tumor biopsy, the two lines differed in their method of in vitro culture establishment with one line (HA-A) grown first in agar culture and then transferred to liquid culture, while the second cell line (HA-L) was established directly from cells grown in liquid culture. Detailed characterization of both cell lines was performed using techniques to analyze their cytogenetic, surface marker, morphology (light and electron microscopy), growth (in agar and in nude mice), and drug sensitivity profiles in comparison to the original tumor biopsy. Our results revealed no significant biologic or genetic difference between the two cell lines established by different culture techniques. Our results suggest that in some cases agar culture prior to liquid culture may be useful in establishment of human tumor cell lines.