ABSTRACT Pollen diffusates, known to be important in pollen-stigma interactions controlling inter-specific incompatibility between Populus deltoides and Populus alba, have been partly characterized and shown to contain more than 20 protein bands by polyacrylamide gel electro-phoresis, at least 4 of these being glycoproteins. Seven fractions had antigenic activity in rabbits and several enzyme activities were also present. Peroxidase and leucine aminopeptidase isoenzymes were detected in the diffusates, demonstrating the extracellular location of these 2 enzymes. Isoenzyme patterns of peroxidase, esterase and acid phosphatase were complex, with some bands common to both species. Localization of acid phosphatase in the intine and esterase in the exine was demonstrated after brief aldehyde fixation and low-temperature embedding in glycol methacrylate. The in tine and exine sites were distinguished by their chemical and structural features. Calcofluor white M2R new proved to be an excellent stain for differentiating the intine. Aniline blue-positive material, probably β-1,3-glucan, is present associated with the intine of many ungerminated as well as germinating grains: production of this material may be a response to damage.