To facilitate studies of cell growth regulation by T3, we developed a variant GC cell line (V-GC) characterized by normal growth in T3-depleted (–T3) medium. The doubling time (dt) of V-GC cells was 28.8 h (–T3) and 28.0 h (+0.2 nM T3), respectively, whereas the dt of the parent GC cells, 24.0 h (+ 0.2 nM T3), increased to more than 100 h (–T3). The dt of V-GC cell was unaffected even by maximal T3 (5 nM). Cell protein (micrograms) per ng DNA increased in GC cells in a T3 concentration-dependent manner, whereas V-GC cell protein was unaffected by T3. GH production appeared partially independent of T3 in V-GC cells. GH production (nanograms per 106/h) in V-GC cells maintained for 3 months in –T3 medium was 3.3- to 4.6-fold greater than that in GC cells after 4 days in –T3 medium (P < 0.001). Addition of T3 resulted in similar maximal GH production in both cell lines. The binding capacity and Ka of nuclear T3 receptors were similar in GC and V-GC cultures, and receptor down-regulation in response to added T3 occurred similarly in both cultures. Lastly, studies employing conditioned medium indicated that T3-independent growth of V-GC cells did not result from production of an autocrine growth factor. Our findings raise the possibility that overexpression of a transacting cell-specific gene-regulating protein that variably affects thyroid hormone-dependent genes may account for the phenotype of the V-GC cultures.