Osteoclast-mediated bone resorption is increased in response to 1,25 dihydroxyvitamin D (1,25[OH],D or calcitriol). Osteopetrosis is a metabolic bone disease characterized by defective, osteoclast-mediated bone resorption, which co-exists with elevated serum 1,25-(OH),D levels in some osteopetrotic children and animals. We examined the effects of high doses of calcitriol on osteoclast number and cytochemistry in both normal and osteopetrotic (0s) rabbits. Calcitriol was continuously infused at doses of 0.5, 2.5, or 25 pg/kg/day via subcutaneously implanted osmotic minipumps for a period of 7 days. Following treatment, the proximal tibia1 metaphyses were processed for histomorphometric and cytochemical analyses. Sections were stained for tartrate-resistant acid phosphatase (TrAP) or acid ATPase (TraATPase). Osteoclasts were significantly reduced in untreated os rabbits compared with age-matched normal littermates between birth and 3 weeks of age (4146% of normal). Whereas most normal osteoclasts (85%) stained heavily for TrAP or TraATPase, less than half of 0s osteoclasts were heavily stained for these acid hydrolases. Infusions of 1,25(OH)zD resulted in elevations of osteoclast numbers in both normal and 0s rabbits, but the number of osteoclasts remained significantly lower in mutants than in normal littermates at any given dose. Calcitriol infusions also resulted in a significant increase in the percentage of 08 osteoclasts staining heavily for TrAP and TraATPase. These results suggest that in response to 1,25(OH)2D normal osteoclasts increase their production of acid hydrolases before increasing cell numbers and that, in spite of high levels of endogenous calcitriol, 0s rabbits can respond to exogenous 1,25(OH)2D as evidenced by increased osteoclast number and cytochemical staining, even though these osteoclasts fail to resorb the excess skeletal matrix. Thus the defect in the mutant skeleton appears to be in the initial cytodifferentiation of osteoclasts, not in their responsiveness to endogenous regulators of bone resorption.