The mouse eye displays unusually rapid intraocular pressure (IOP) responses to topically applied drugs as measured by the invasive servo-null micropipette system (SNMS). To learn if the time course reflected rapid drug transfer across the thin mouse cornea and sclera, we monitored a different parameter, pupillary size, following topical application of droplets containing 40 microM (0.073 microg) carbachol. No miosis developed from this low carbachol concentration unless the cornea was impaled with an exploring micropipette as used in the SNMS. We also compared the mouse IOP response to several purinergic drugs, measured by the invasive SNMS and non-invasive pneumotonometry. Responses to the previously studied non-selective adenosine-receptor (AR) agonist adenosine, the A(3)-selective agonist Cl-IB-MECA and the A(3)-selective antagonist MRS 1191 were all enhanced to varying degrees, in time and magnitude, by corneal impalement. We conclude that the thin ocular coats of the mouse eye actually present a substantial barrier to drug penetration. Corneal impalement with even fine-tipped micropipettes can significantly enhance entry of topically-applied drugs into the mouse aqueous humor, reflecting either direct diffusion around the tip or a more complex impalement-triggered change in ocular barrier properties. Comparison of invasive and non-invasive measurement methods can document drug efficacy at intraocular target sites even if topical drug penetration is too slow to manifest convincing physiologic effects in intact eyes.