During contractions, there is a net efflux of phosphate from skeletal muscle, likely due to an elevated intracellular inorganic phosphate (P_i) concentration. Over time, contracting muscle could incur a substantial phosphate deficit unless P_i uptake rates were increased during contractions. We used the perfused rat hindquarter preparation to assess [³²P]P_i uptake rates in muscles at rest or over a range of energy expenditures during contractions at 0.5, 3, or 5 Hz for 30 min. P_i uptake rates were reduced during contractions in a pattern that was dependent upon contraction frequency and fiber type. In soleus and red gastrocnemius, [³²P]P_i uptake rates declined by ~25% at 0.5 Hz and 50-60% at 3 and 5 Hz. Uptake rates in white gastrocnemius decreased by 65-75% at all three stimulation frequencies. These reductions in P_i uptake are not likely confounded by changes in precursor [³²P]P_i specific activity in the interstitium. In soleus and red gastrocnemius, declines in P_i uptake rates were related to energy expenditure over the contraction duration. These data imply that P_i uptake in skeletal muscle is acutely modulated during contractions and that decreases in P_i uptake rates, in combination with expected increases in P_i efflux, exacerbate the net loss of phosphate from the cell. Enhanced uptake of P_i must subsequently occur since skeletal muscle typically maintains a relatively constant total phosphate pool.