During the last 15 years, the methods proposed to study the regulation of cholinergic transmission in vivo have required the labeling of the acetylcholine (ACh) and choline stored in neurons by injecting labeled precursors of ACh, and by measuring simultaneously the changes with time in the specific activities of choline and ACh (Cheney et al., 1974, 1975a; Costa et al. 1975; Dross and Kewitz, 1966; Hanin et al., 1973; Jenden et al., 1974; Racagni et al., 1974; Saelens et al., 1973; Schuberth et al., 1969; Zsilla et al., 1977). In addition to these isotopic methods, various nonisotopic approaches have been suggested to estimate the fractional rate constant of ACh efflux (Bartholmi et al., 1976; Celesia and Jasper, 1966; Hemsworth and Neal, 1968; Mitchell, 1960; Pepeu et al, 1975; Smith, 1972; Szerb, 1975; Yaksh and Yamamura, 1975). Some of the nonisotopic methods have used drugs to instantaneously inhibit synthesis, metabolism, or transport of ACh, its precursor, or metabolites (Costa and Neff, 1966). A flaw common to all the methods involving the use of inhibitors derives from the widely spread assumption that drugs are so specific that, even in high doses, they selectively inhibit a given metabolic step. Obviously, such specificity is exceptional; more often, drugs that inhibit ACh metabolism exert collateral actions on other processes that directly or indirectly regulate a number of other metabolic steps.