The possibility that acidosis inhibits lipolysis indirectly by causing ionic shifts or by favouring the accumulation of an inhibitor has been tested in isolated fat cells. Lipolysis induced by 3 muM noradrenaline (NA) was inhibited by 40-60% and that induced by 1 mM theophylline (THEO) by about 75% when the pH was reduced to 6.6. Lipolysis induced by NA + THEO was inhibited by 20-30%. Changing the concentration of Ca++ or Mg++ did not alter the degree of inhibition. Reducing the K+-ion concentration enhanced the inhibitory effect of low pH on lipolysis induced by NA or NA + THEO, whereas cyclic AMP accumulation was uninfluenced. Omitting glucose from the incubation medium caused a slight enhancement of pH-induced inhibition of lipolysis (from 60 to 70%, p less than 0.01). Reducing the concentration of albumin, which binds inhibitory substances such as FFA, reduced lipolysis more at normal than at reduced pH. At high FFA/albumin ratios (5 or above) lipolysis was similar at normal and reduced pH. The antilipolytic effect of decreased pH was equally pronounced in perifused fat cells, where inhibitory substances are not allowed to accumulate. Our results suggest that the antilipolytic effect of acidosis is mainly a direct effect of the increase in H+ ion concentration. The inhibitory effect of acidosis on various responses to beta-adrenoceptor stimulation may be caused by a decreased formation of cyclic AMP in turn caused directly by the decrease in pH.