In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, mi crovillus-like cell protrusion. Formation of this ≃3-μm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt-) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mr- gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated rot+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2- 3 x 106 fertilization tubules/μg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt- gametes between the two flagella, the site of the mt- mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt-gametes.