180 pitfalls of relying solely upon pharmacological data, we supplemented behavioral assays with other kinds of tests, such as Western blots, immunofluorescence, and EIA when available. In all cases, the additional tests confirmed the data we originally obtained using pharmacological inhibitors. Taken together, our data indicate that a number of enzymes are involved in GTP signaling. However, further experimentation is needed to discern the interrelationships between enzymes in the pathway and to answer the myriad questions that remain. For example, are guanylyl cyclase and NOS truly activated by calcium in Tetrahymena? Would calcium chelation abolish their activity? Is there a soluble guanylyl cyclase in Tetrahymena? What NOS isoforms do Tetrahymena possess? Is tyrosine phosphory- lation required for either NOS or guanylyl cyclase activity? Are any additional proteins, particularly ciliary proteins, involved in GTP signaling? Are all of the components of the pathway located in the cilia? These and many other questions remain to be answered. One question we have begun to answer is whether intracellular calcium is required for tyrosine kinase activity. Recent immunofluorescence shows no difference in phosphotyrosine levels between GTP-treated cells and GTP-treated cells that had been pretreated with BAPTA-AM (unpublished data). Similarly, calcineurin (PP2B) does not appear to be involved in GTP signaling, as ascomycin treatment had no effect on GTP avoidance (unpublished data). Endothall, a PP2A inhibitor, did inhibit avoidance (unpublished observation), consistent with observations by Deckman and Pennock [33] that PP2A is involved in ciliary reversal. Further experimenta- tion will help us determine the interrelationship of pathway components and better establish the mechanism by which chemorepellents signal in Tetrahymena. 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