The general phosphate need in mammalian cells is accommodated by members of the PiT-family (SLC20), which use either Na⁺ - or H⁺ - to mediate inorganic phosphate (P_i) symport. The mammalian PiT-paralogs, PiT1 and PiT2, are Na⁺-dependent P_i (NaP_i) transporters and exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes using ³²P_i as traceable P_i source. For PiT1 the Michaelis-Menten constant for P_i, Km_Pi, was determined to 322.5 ±124.5 µM. PiT2 was here analyzed for the first time and showed positive cooperativity in P_i uptake with a K_0.5Pi of 163.5 ±39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pHs and displayed similar Na⁺-dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations, Ca²⁺ and Mg²⁺, revealed that Ca²⁺ was important - but not critical - for NaP_i transport function of the PiT proteins. To gain insight into the NaP_i cotransport function, PiT2 and a PiT2 P_i-transport knockout mutant were analyzed using 22Na⁺ as traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake media and also when P_i transport function was knocked out. This is the first time de-coupling of P_i from Na⁺ transport has been demonstrated for a PiT-family member. Moreover, the results imply that putative transmembranic amino acids, E₅₅ and E₅₇₅, are responsible for linking P_i import to Na⁺ transport in PiT2.