The anion dependence of [3H]bumetanide binding and 22Na+ transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter was studied in acinar basolateral membrane vesicles (BLMVs). Cl-, Br- and NO3- have a biphasic effect on binding consistent with the presence of two anion sites associated with the bumetanide binding event, a high-affinity stimulatory site and a lower-affinity inhibitory site. We show that formate shares only the stimulatory site and SO4(2-) only the inhibitory site. The initial rate of [3H]bumetanide binding was stimulated by formate or low [Cl-] and inhibited by SO4(2-) or high [Cl-], but the rate of [3H]bumetanide dissociation was not affected by the presence of these anions in the dissociation medium. However, when [3H]bumetanide was bound to BLMVs in the presence of formate its rate of dissociation was more than four times faster than when binding took place in the presence of Cl-. These observations indicate that the binding of bumetanide and the stimulatory anion are ordered such that the anion must necessarily bind first and subsequently cannot dissociate until after bumetanide dissociates. In zero-trans-flux experiments, extravesicular SO4(2-) and formate had no effect on 22Na+ transport via the co-transporter [Turner and George (1988) J. Membr. Biol. 102, 71-77]. Thus neither of the anion sites associated with bumetanide binding is a Cl- transport site. However, we show here that SO4(2-) inhibits transport when present in the intravesicular space. Since the BLMV preparation is predominantly oriented cytosolic-side-in, this observation indicates the existence of an inhibitory cytosolic anion modifier site. Our data suggest that this site is identical to the inhibitory anion site associated with bumetanide binding.