A simian virus 40 (SV40)-transformed line of human parapharyngeal cells (SV-TGo) was cultured in semisolid agar to determine its ability to grow in the absence of an anchoring substratum and to evaluate any phenotypic changes that might have resulted during the isolation of sublines specifically selected for anchorage independence. After 2--3 weeks and 14--15 population doublings in culture, SV-TGo plated with over 1,000% higher efficiency than negative controls (F2408 cells). Sublines, 0.3--2.0 mm in diameter, were isolated and transferred to Leighton tubes in which they underwent an additional 0--7 divisions before senescence after 39--44 total population doublings. Subline phenotype was identical to the original parental phenotype, including epithelioid morphology, organized pattern of growth, extreme sensitivity to density-dependent inhibition of growth, and continuous production of infectious SV40 as detected by the combined tests of cocultivation and direct isolation. Limited division potential was within the range observed for the parental line. The ability to grow in agar without identifiable phenotypic changes was therefore confirmed for this line of SV40-transformed human epithelioid cells.