Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 126.96.36.199) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 188.8.131.52), polygalacturonase (EC 184.108.40.206), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.