Within the renal cortical collecting duct (CCD), transepithelial Na⁺ absorption and K⁺ secretion are linked to basolateral Na⁺-K⁺-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na⁺-K⁺-ATPase-mediated ⁸⁶rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na⁺ transport. Multiple CCDs (~6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37°C for 10 min (±1-2.5 mM ouabain or 10 or 100 µM Schering-28080, an inhibitor of H⁺-K⁺-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of ⁸⁶Rb (±inhibitors). After 1 min at 37°C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na⁺-K⁺ pump activity, increased ~3-fold during the 1st 8 wk of postnatal life (P < 0.03). The ~2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 ± 0.45 to 5.02 ± 0.32 pmol · min¹ · mm¹) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 ± 0.53 pmol · min¹ · mm¹ in adult animals (P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H⁺-K⁺-ATPase is absent at the basolateral membrane. Na⁺-K⁺-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [-³²P]ATP under maximum velocity (V_max) conditions, also increased in the differentiating CCD (from 316.2 ± 44.4 pmol · h¹ · mm¹ at 2 wk to 555.9 ± 105.1 at 4 wk to 789.7 ± 145.0 at 6 wk; r = 1.0 by linear regression analysis; P < 0.005). The parallel ~2.5-fold increases in Na⁺-K⁺-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na⁺-K⁺-ATPase activity in the CCD remain to be defined.