Topical administration of 50 microliter of 1% Na3VO4 caused a significant fall in intraocular pressure (IOP) in the rabbit eye at 90 min. Assay of ATPase of the iris and ciliary body in vitro at 90 min posttreatment showed no differences between control and treated (Na+, K+)ATPase, ouabain sensitive ATPase or vanadate sensitive ATPase. Accumulation of 48V-labelled orthovanadate in iris and ciliary body reached a plateau of 12 pmoles/mg dry tissue weight 4 hr after a single 25-microliter topical dose of 1% orthovanadate. In vitro inhibition of Na+ sensitive ATPase by sodium metavanadate had an IC50 of 1.8 mM, whereas a 1.8-fold stimulation of adenylate cyclase in iris-ciliary body (ICB) membranes in vitro occurred at 10 mM but not at 10 microM Na metavanadate. These results indicate that the vanadate content of the iris and ciliary body at the time of lowered intraocular pressure is too small to inhibit a significant fraction (greater than 10%) of (Na+, K+)ATPase, or cause a significant stimulation of adenylate cyclase, and that other cellular mechanisms are likely to be involved in the ocular hypotensive response to vanadate.