This study explores the role of the calmodulin- and Ca²⁺-sensitive phosphatase, calcineurin A, in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin A and A cDNA from a rabbit osteoclast library. Sequence analysis revealed a ~95% and 86% homology between the amino acid and nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin A mRNA in freshly isolated rat osteoclasts. Semi-quantitative analysis of staining intensity revealed no significant difference in calcineurin A expression in cells treated with vehicle versus those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10^-7 M) and FK506 (5 x 10^-9 and 5 x 10^-7 M). We then constructed a fusion protein comprising calcineurin A and TAT, a 12-amino acid-long Arg-rich sequence of the HIV protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Likewise, unfolding of the TAT-calcineurin A fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin A and anti-TAT antibodies. Pit assays performed with TAT-calcineurin A-positive osteoclasts revealed a concentration-dependent (10 to 200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinnin did not produce significant effects on bone resorption or cell number. The study suggests that (a) the 61 kD protein phosphatase, calcineurin A, can be effectively tranduced into osteoclasts using the TAT-based approach, and (b) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.