The DNA synthesis (S) phase of cultured GC cells, a clonal rat pituitary tumor cell line, is characterized by increases in nuclear receptors for T3 and glucocorticoid (G) hormones. However, this increased receptor abundance appears functionally dissociated from the GH gene since GH messenger RNA (mRNA) synthesis is decreased in S-phase cells. We have now examined a putative structural correlate of this dissociation by measuring the abundance of T3 and G receptors and the GH gene in the nuclear matrix (NM)/scaffold fraction. NM of control and S-phase cultures both contained 30-50% of G receptors. Thirty to 50% of T3 receptors were also localized to NM of asynchronous cultures, but T3 receptor abundance (femtomoles per 100 micrograms of protein) was significantly decreased in NM of S-phase cultures: Exp 1 (control, 140 +/- 6.2; S-phase, 56.5 +/- 0.8; Exp 2 (control, 170 +/- 12; S-phase, 105 +/- 2.4). Nuclear scaffolds were digested with restriction enzymes to solubilize DNA loop structures and probed with GH complementary DNA (cDNA) to examine the distribution of transcribed regions of the GH gene. EcoR1 digestion resulted in an 11 kilobase fragment including the reported regulatory sequences for T3 receptors. No specific differences in GH gene localization to nuclear scaffold were observed between asynchronous and S-phase cultures or in different hormonal states. Thus, GH gene localization to nuclear scaffolds was not correlated with changes in transcription induced by T3 and G hormones or position in the cell cycle. These studies suggest that decreased concentration of T3 receptors in the NM fraction of S-phase cultures may be a structural correlate for cell cycle regulation of T3 receptor function.