Gametes of opposite mating type (mt (+) and mt (-)) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using (35)S-labeled flagella and the isolated mt (-)agglutination factor. It is shown that not only isolated flagella, but also the mt (-)agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt (-)agglutination factor in determining the sexual agglutinability of mt (-)gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt (-)agglutination factor can be completely inactivated.