Technical Abstract: Thyroid hormones are galactopoietic and appear to assist in establishing the mammary gland¿s metabolic priority during lactation. Expression patterns for genes that can alter tissue sensitivity to thyroid hormones and thyroid hormone activity were evaluated in the mammary gland and liver of Holstein cows at 53, 35, 20 and 7 days before expected parturition and 14 and 90 days into the subsequent lactation. Transcripts for the three isoforms of iodothyronine deiodinase, type I (DIO1), type II (DIO2) and type III (DIO3) and transcripts for the thyroid hormone receptors alpha1 (TRa1), alpha2 (TRa2) and beta1 (TRb1) were evaluated. Tissues for this purpose were obtained from 3 to 5 euthanized cows per physiological state. The DIO3 is a 5-deiodinase that produces inactive iodothyronine metabolites, whereas DIO1 and DIO2 generate the active thyroid hormone, triiodthyronine, from the relatively inactive precursor, thyroxine. Low copy numbers of DIO3 transcripts were present in mammary gland and liver during all physiological conditions. DIO2 was the predominant isoform expressed in mammary gland and DIO1was the predominant isoform expressed in liver. Quantity of DIO1 mRNA in liver tissues did not differ (P > 0.1) with physiological state, but tended to be lowest during lactation. Quantity of DIO2 mRNA in mammary gland increased during lactation (P < 0.05), with copy numbers at 90 d of lactation 6-fold greater than at 35 and 20 d prepartum. When ratios of DIO2/DIO3 were evaluated, the increase was even more pronounced (>100-fold). Quantity of TRß1 mRNA in mammary gland increased with onset of lactation, whereas TRa1 and TRa2 did not vary with physiological state (P > 0.1). Conversely, quantity of retinoic acid receptor alpha (RXRa) mRNA decreased during late gestation to low levels during early lactation. Data suggest that increased expression of mammary TRß1 and DIO2, and decreased RXRa, provide a mechanism to increase thyroid hormone activity within the mammary gland during lactation.