The growth characteristics of multicellular spheroids, derived from human melanoma xenografts and cultivated in liquid-overlay culture, were studied and compared with those of the parent tumours. Six of the seven melanomas investigated formed spheroids, which grew exponentially up to a volume of 1-2 x lo7 pm3 (a diameter of 270-340 pm) before the growth rate tapered off. The morphology of the spheroids varied considerably among the melanomas; some spheroids grew as densely packed, spherical structures of cells whereas others were loosely packed and showed an irregular shape. Central necrosis developed when the spheroids attained a diameter of 150-200 ,urn. The histological and cytological appearance of the spheroids was remarkably similar to that of the parent xenograft in five of the six cases. The sixth melanoma contained two subpopulations with distinctly different DNA content, one of which was predominant in the spheroids, the other in the tumours. This gave rise to clear histological and cytological differences. The volume-doubling time of the spheroids during the exponential growth phase ranged from 1.7 k 0.2 to 2.7 0.4 days and the fraction of cells in S from 13 & 1 to 28 & 2%. The volume-doubling time decreased with increasing fraction of cells in S, indicating that the differences in growth rate were due mainly to differences in the growth fraction or to differences in the duration of G,. The spheroid volume-doubling times did not correlate with those of the parent xenografts (T, = 4.2-22.5 days at V = 200 mm3), possibly because the cell loss factors of the xenografts were large and varied among the melanomas. The fractions of cells in G,/G, S and G, + M in the spheroids and the xenografts did not correlate either, but were found to be within the same narrow ranges in the spheroids and the xenografts4.e. 50-80% (G,/G,,), 10-30% (S) and 10-20% (G, + M).Multicellular spheroids are an in v i m tumour model system representing an intermediate level of complexity between monolayer cell cultures and solid tumours (Sutherland el al., 1970;Sutherland & Durand, 1976). Spheroids resemble tumours in that the cells grow in a three-dimensional structure which facilitates intercellular communication (Durand &