Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P_i) and the high energy organic pools of ATP and phosphocreatine (PCr). At steady state, the uptake of P_i into the cell must equal the rate of efflux, which is expected to be a function of intracellular P_i concentration. We measured [³²P]P_i uptake rates in different muscle fiber types to determine if they are proportional to cellular P_i content. P_i uptake rates in isolated perfused rat hindlimb muscles were linear over time and greatest in soleus (2.42 ± 0.17 µmol/g/h), lower in red gastrocnemius (1.31 ± 0.11 µmol/g/h), and least in white gastrocnemius (0.49 ± 0.06 µmol/g/h). Reasonably similar rates were obtained in vivo. P_i uptake rates at plasma P_i concentrations of 0.3-1.7 mM confirm that the P_i uptake process is nearly saturated at normal plasma P_i levels. P_i uptake rate correlated with cellular P_i content (r = 0.99), but varied inversely with total phosphate content. Sodium/phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius was similar to each other and 7- to 8-fold greater than Pit-1 expression in white gastrocnemius. Because PiT-1 expression pattern did not match the pattern of P_i uptake across fiber types implies that other factors are involved in regulating P_i uptake in skeletal muscle. Further, fractional turnover of the cellular P_i pool (0.67, 0.57, and 0.33 h^-1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P_i, likely due to differences in the resistance to P_i efflux from the fiber.