1. Arachidonic acid (AA) exerts multiple physiological and pathophysiological effects in the brain. By continuously measuring the intracellular pH (pH(i)) and Ca2+ levels ([Ca2+]i) in primary cultured rat cerebellar granule cells, we have found, for the first time, that 20 min treatment with 10 microM AA resulted in marked increases in Ca2+ and H+ levels in both the cytosol and nucleus. 2. A much higher concentration (40 mM) of another weak acid, propionic acid, was needed to induce a similar change in pH(i). The [Ca2+]i increase was probably caused by AA-induced activation of Ni2+-sensitive cationic channels, but did not involve NMDA channels or the Na+-Ca2+ exchanger. 3. AA-induced acidosis occurs by a different mechanism involving predominantly the passive diffusion of the un-ionized form of AA, rather than a protein carrier, as proposed by Kamp & Hamilton for fatty acids (FAs) in artificial phospholipid bilayers (the 'flip-flop' model). The following results, which are similar to those observed in lipid bilayers, support this conclusion: (1) FAs containing a -COOH group (AA, linoleic acid, alpha-linolenic acid, and docosahexaenoic acid) induced intracellular acidosis, whereas a FA with a -COOCH3 group (AA methyl ester) had little effect on pH(i), (2) a FA amine, tetradecylamine, induced intracellular alkalosis, and (3) the AA-/FA-induced pH(i) changes were reversed by bovine serum albumin. 4. Further evidence in support of a passive diffusion model, rather than a membrane protein carrier, is that: (1) there was a linear relationship between the initial rate of acid flux and the concentration of AA (2-100 microM), (2) acidosis was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, a potent inhibitor of the plasma membrane FA carrier protein, and (3) the involvement of most known H+-related membrane carriers and H+ conductance has been ruled out. 5. Since AA can be released under both physiological and pathophysiological conditions, the possible significance of the AA-evoked increases in H+ and Ca2+ in both the cytoplasm and nucleoplasm is discussed.