Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.