We investigated the possibility that hypothyroidism, like starvation, leads to a major disaggregation of polyribosomes and redistribution of mRNA to the nonpolysomal mRNA particle (mRNP) pool. The analysis involved the use of a translational assay of hepatic RNA obtained from bound and free polysomes and the subribosomal fractions (mRNP) of euthyroid and hypothyroid rats. Two-dimensional gel electrophoretograms of the in vitro synthesized 35S-labeled proteins revealed distinct protein patterns associated with each subcellular compartment. Of the 18 quantified T3-responsive sequences, the activities of 7 were preferentially associated with bound polysomes, 9 with free polysomes, and 2 with subribosomal mRNP in euthyroid rat livers. No major redistribution occurred in the transition from the euthyroid to the hypothyroid state. The distribution of mRNA activity of 1 T3-responsive sequence, that coding for Spot 14 (mRNAS14), was compared with the distribution of hybridizable mRNA, as determined with the use of a specific cDNA probe. In the euthyroid state, both techniques showed that approximately 70% of the total hepatic mRNAS14 was associated with free polysomes and 30% was associated with subribosomal complexes. A similar distribution was characteristic of the hypothyroid state. These findings demonstrate that T3-responsive sequences are found in all three mRNA compartments and that thyroid hormone action is not typically associated with a redistribution of mRNA among subcellular pools.