The significance of low pH-induced stimulation of osteoclastic bone resorption has recently been questioned following the finding that embryonic chick osteoclasts were only weakly stimulated by extremely low pH (6.5) and that the effect was transient, apparently due to cytotoxicity. Although low pH in the range 6.8-7.2 is known to stimulate rat osteoclasts over 24 h, the long-term effects of low pH on mammalian osteoclasts are not known. We have therefore conducted time-course studies over 72 h on the effect of pH in the range 6.3-7.3 on bone resorption and cytotoxicity in both rat and chick osteoclasts. In neonatal rat osteoclasts, lowering extracellular pH produced a powerful and significant stimulation of resorption over 24 h. Detailed analysis of the resorption focus revealed that this was due mainly to a higher proportion of active osteoclasts at lower pH. In addition, osteoclasts excavated slightly larger pits at low pH. Stimulation was no longer significant at 72 h, however, due to a pH-dependent slowing of resorption at acid pH associated 1) with cytotoxicity primarily of nonosteoclastic cells and 2) with an acceleration of bone resorption after 24 h at more alkaline pH. Resorption stimulated by low pH was associated with the formation of actin-rich "clear zones" within the osteoclast. Chick osteoclasts were less sensitive to low pH than rat osteoclasts but nonetheless showed a consistently higher level of resorption at low pH over 24-72 h. These results suggest that protons play an important regulatory role in neonatal rat osteoclasts, and stimulate the formation of clear zones. The lower sensitivity of the chick osteoclast to acid pH may be due to a species difference or the chick osteoclast's higher basal level of resorption.