ABSTRACTThis study investigates increases in cellular calcium concentration during sexual reproduction of the heterothallic water mold,Achlya ambisexualis.Cellular localization of free calcium was monitored with the fluorescent calcium-binding antibiotic, chlorotetracycline (CTC). Other than the generalized fluorescence in the hyphal apex associated with vegetative growth, CTC-Ca fluorescence was first observed in the male strain during antheridial branch production. It became restricted to antheridia after septal delimitation and persisted until fertilization. Bright CTC-Ca fluorescence was not observed in the female strain until after antheridial contact with the oogonial initial and septal delimitation of the oogonium. The fluorescence continued through subsequent stages of development, including post-fertilized oospores. This study demonstrates that an increase in Ca+2concentration in the male strain is associated with antheridial branch formation, vectorial growth toward the oogonial initial and fertilization. In the female strain, development of the oogonial cytoplasm after septal delimitation, oosphere formation, and oospore maturation correlate with calcium increase. These results target key events in sexual reproduction for which the precise cause and effect relationship between changes in calcium concentration and morphogenesis can be investigated.