In addition to its normal synthesis in pituitary and placenta for production of the four glycoprotein hormones (GPH), the free α-subunit is produced by a variety of tumors and tumor-derived cell lines. It has long been used clinically as a tumor marker, but the question remains as to whether ectopic production of GPHα represents the random and chance activation of the hormone gene during cancer development or whether GPHα may contribute directly or indirectly to the biology of the tumor. One characteristic of tumorigenic cells in culture is their ability to proliferate in an anchorage-independent way. Data are presented in the following paper to show that the cloning efficiency of HeLa cervical carcinoma cells in soft agar is directly correlated with their production of the GPH α-subunit. HeLa variants that differ over 400-fold in their production of GPHα similarly exhibited a marked difference in their ability to form colonies in 0.3% noble agar. When HeLa SR3 cells (a variant that produces GPHα at high levels) was stably transfected with a vector producing the antisense strand of GPHα cDNA, the synthesis of the GPH α-subunit was reduced in these cells as was their cloning efficiency in soft agar. Similarly, when HeLa A5F cells (a variant producing little or no GPHα) were stably transfected with an α-subunit expression vector, the production of GPHα was increased significantly in concert with an increased ability to form colonies in 0.3% noble agar. Somatic cell hybrids between HeLa SR3 and HeLa A5F exhibited intermediate levels of GPHα gene expression and colony formation in soft agar compared to the parental strains. These data suggest that some parameters of the tumorigenic phenotype, such as anchorage-independent growth, are responsive to, or are dependent upon, the production of free α-subunit.