It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4  · cm², respectively. The apical membrane potential was 43.9 ± 1.7 mV, and the ratio of apical to basolateral membrane resistance (R_A/R_B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC₅₀ of ~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM) depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreased R_A/R_B by a factor of 3. The addition of adenosine to either side (100 µM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca²⁺ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca²⁺ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 ± 3 µl · cm² · h¹ (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y₂ purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca²⁺-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.