White adipocytes are an important target tissue for thyroid hormones, which enter the cells largely by a System L1-type amino acid transporter en route to exerting genomic actions in the nucleus (Ritchie et al., 2001). We are investigating the regulation of this process using differentiated 3T3-L1 adipocytes and L-[³H]phenylalanine as a transportable tracer for System L1 activity. 3T3-L1 cells were cultured at 37^oC, 5% CO₂ in Dulbecco’s Modified Eagles Medium (high-glucose) with 10% serum and 1% antibiotic/antimycotic solution; they were differentiated into adipocytes using 1 µg.ml^-1 insulin, 5 µM isobutylmethylxanthine and 100 pM dexamethasone (Hyde et al., 2001) . Phenylalanine uptake into 3T3-L1 cells was saturable (K_m of 31 µM, V_max of 2.6 ± 0.5 nmol. mg protein^-1. min^-1; mean ± sem, n=6) and inhibited by the synthetic System L substrate 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH):- 5 µM phenylalanine uptake (0.465 ± 0.065 nmol. mg protein^-1. min^-1, n=12) was reduced by 98% in presence of 10 mM BCH. Phenylalanine uptake was competitively inhibited by T₃ (K_i of 1.2 µM) but not by Triac (a T₃ derivative lacking the α-amino grouping) and was also blocked by leucine and rT₃ in a manner consistent with expected substrate interactions for the System L1 transporter (Ritchie et al., 1999; Friesema et al., 2001). We detected mRNA for LAT1 (the catalytic subunit of System L1) in 3T3-L1 cells using RT-PCR. Efflux of preloaded L-[³H]phenylalanine from 3T3-L1 adipocytes was trans-stimulated by external leucine (up to 20-times stimulation of basal efflux), demonstrating the obligatory exchange mechanism of System L1 transport (Meier et al., 2002). T₃ has a relatively low V_max for transport by System L (Ritchie et al., 1999) and was unable to significantly trans-stimulate L-[³H]phenylalanine efflux at 10 µM (a concentration approaching the limit of T₃ solubility in culture medium), although it did inhibit the trans-stimulatory effect of 10 µM leucine. System L transport activity was reduced by 40% in 3T3-L1 cells cultured in thyroid-hormone depleted (charcoal-treated) medium for 24 hours. The present results confirm a strong competitive interaction between iodothyronines (T₃, rT₃) and amino acids for transport by System L1 in adipocytes. Down-regulation of iodothyronine uptake by adipocytes in the hypothyroid state, as also reported previously (Ritchie et al., 2001), should reduce their catabolism and help conserve hormone and iodine availability to other tissues.