Using rat 13762NF mammary tumor cell clones of varying spontaneous metastatic potentials and biochemical properties and known phenotypic stabilities we studied the relationship between cell colony growth in a clonogenic assay and the biological and biochemical properties of cells derived from different cell colonies. The spontaneous metastatic potential of im vivo or in vitro grown 13762NF tumor cells was not related to their in vitro cloning efficiencies; cells of both low and high metastatic potential formed colonies of various sizes and shapes during 14 days of growth in 0.3% or 0.6% semisolid agarose. A highly metastatic cell done of relatively low growth potential in agarose was examined further. Individual tumor cell colonies derived from this cell done were removed from agarose and their properties determined. Cells from small (100-μm-diameter) or large (500-μm-diameter) agarose colonies had similar self-renewal capadties in agarose and formed variously sized cell colonies when replated in agarose medium. Metastatic potential, drug sensitivity parameters, and expression of a high Mr mucin-like glycoprotein antigen and type IV collagenolytic activity known to be associated with spontaneous metastasis of 13762NF tumor cells were dissimilar in cells from different colonies, and these characteristics were independent of original tumor cell colony size in agarose. In contrast, the expression of cell surface proteins of Mr 300,000 were similar among cells derived from different agarose colonies. The data indicate that heterogeneity exists in the ability of 13762NF adenocarcinoma cells of different biochemical and metastatic potentials and drug sensitivities to grow in semisolid agarose. In addition, the cells that grow in agarose to form detectable colonies (50 cells) are not necessarily those with a high potential of metastasizing spontaneously to distant sites. © 1988, American Association for Cancer Research. All rights reserved.