Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT-PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any. RANKL mRNA was detected in two of eight rat and one of four human samples. Treatment of osteoclasts with soluble RANKL resulted in translocation of NF-κB to the nucleus and elevation of cytosolic and nuclear calcium levels. We have shown that RANK is highly expressed in mature osteoclasts and that its stimulation by RANKL results in activation of NF-κB and calcium signalling.