Deletion of the twinfilin gene from budding yeast results in defects in the organization of cortical actin patches. Together with certain cofilin and profilin mutations, twinfilin deletion leads to synthetic lethality. Inactivation of twinfilin in flies led to severe defects in a number of actin-dependent processes. These include e.g. abnormal bristle and eye morphology as well as defects in axonal growth in the brain and border cell migration in the ovary. Twinfilins also play an important role in actin-dependent processes in Pamidronate disodium pentahydrate cultured mammalian cells, because depletion of either twinfilin-1 or twinfilin-2a from HeLa cells by RNAi resulted in defects in endocytosis and depletion of twinfilin-2a from SH-SY5Y cells restrained neurite outgrowth. Furthermore, depletion of twinfilin-1 results in defects in invasive motility of lymphoma cells, whereas up-regulation of twinfilin-1 promoted cardiac hypertrophy. However, despite the wealth of data on cultured mammalian cells, the role of mammalian twinfilin isoforms in animal models has not been reported. As a first step towards understanding the role of twinfilins in mammalian development and physiology, we generated twinfilin2a knockout mice. Surprisingly, although studies on cultured cells indicated that this protein plays an important role in endocytosis and neurite outgrowth, ablation of twinfilin-2a did not lead to obvious defects in mouse development. These data suggest that in mice twinfilin-2a and twinfilin-1 may have redundant roles in promoting actin dynamics in non-muscle cells. Western blot analysis using isoform-specific antibodies revealed that twinfilin-2 protein was present at detectable levels in all tissues of the analyzed Timosaponin-BII wild-type mouse, whereas twinfilin-2 protein was undetectable in all knockout mouse tissues except heart and skeletal muscle. It is important to note, that this antibody does not distinguish between twinfilin-2a and twnfilin-2b proteins. In heart extracts, a slightly smaller protein, representing twinfilin-2b, was detected. In skeletal muscle extracts, three proteins with slightly different molecular weights were detected, most likely resulting from post-translational modification of twinfilin-2b. The smaller protein from wild-type and knockout mouse liver extracts is most likely a result of unspecific binding of the twinfilin-2 antibody.