alpha 2-Macroglobulin (alpha 2-M) is known as a wide-spectrum proteinase inhibitor and to bind covalently certain growth factors. We have previously characterized tumor-associated alpha 2-M synthesized and secreted by human tumor cell lines. Of the cell lines studied, the melanoma cell line HMB-2 produced the largest amount of this glycoprotein. Immunofluorescence staining of cultured HMB-2 cells suggested that the cell population is heterogeneous with respect to alpha 2-M production. We have now isolated clones from the parental HMB-2 cells and characterized eight representative ones in detail. They varied considerably in the quantity of alpha 2-M secreted, from 4.2 to 46.5% of total protein. No relationship between the production of alpha 2-M by these clones and their pigmentation or tumorigenicity in nude mice was found. However, statistically there was a strong correlation between the modal chromosome number and population doubling time (r2 = 0.88, P less than 0.001) and also between the modal chromosome number and alpha 2-M production (r2 = 0.73, P less than 0.01). The growth rate of the clones correlated with the level of alpha 2-M in culture medium (r2 = 0.69, P less than 0.01). Clones with lower alpha 2-M production had a proportionally shorter population doubling time than the clones with intermediate or high production. Northern hybridization indicated quantitative variation in the alpha 2-M mRNA expressed by the parental cells and the clones, that was comparable but not identical with the quantity of alpha 2-M in the respective culture media; both parental cells and clones expressed platelet-derived growth factor A-chain mRNAs with little difference in levels. Serum-free medium from low alpha 2-M producer clones stimulated normal stationary fibroblasts significantly more than clones producing intermediate or high amounts of alpha 2-M. alpha 2-M decreased and anti-alpha 2-M IgG increased the stimulation. These results suggest that production of tumor-associated alpha 2-M is related to both autocrine and paracrine growth-stimulating activity of the tumor cells.