This study investigates the mechamsms involved in the regulatory volume decrease (RVD) in ZR-75-1 epithelial-derived human breast cancer cells. Cell volume changes were measured during osmotic shock using video imaging. In HEPES-buffered hypotonic solutions no RVD was observed; however, RVD was observed in HCO3 --buffered hypotonic solutions. Inhibition of RVD by 10 μM tamoxifen and 100 μM DIDS (inhibitors of volume-regulated anion channels; VRAC) and 2 mM TEA+ (inhibitor of K+ channels) indicates a role for these channels. In HCO3 --buffered Cl--free solutions RVD was partially abolished indicating that HCO3 - efflux can support RVD but also may have another role. Further experiments investigated whether HCO3 - assists in the accumulation of Cl- via Cl--HCO3 - exchange. Regulatory volume increase (RVI) was also HCO3 --dependent and was inhibited by 500 μM DIDS and 10 μM 5-(N,N-dimethyl)-amiloride (DMA) indicating a role for coupled Cl--HCO3 - and Na+-H+exchange. Finally, in the presence of 10 μM DMA, RVD was partially inhibited providing further evidence for a role of Cl--HCO3 - exchange. Thus RVD in ZR-75-1 cells involves the activation of VRAC and K+ channels. RVD is HCO3 --dependent and HCO3 - efflux through VRAC appears to contribute directly to RVD. HCO3 -, however, also has another role in facilitating Cl- accumulation via Cl--HCO3 - exchange.