One of the biggest challenges of in vivo gene therapy is the lack of selective vector systems for a defined therapy-relevant cell population. Here we present a second generation of designed ankyrin repeat protein (DARPin)-targeted AAVs (DART-AAVs) exhibiting improved DARPin display and gene transfer activity. Insertion of the human CD8-specific DARPin 63A4 into the GH2/3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in vector stocks with unimpaired activities as compared to unmodified serotypes. The capsid core structure was unaltered with the DARPins protruding from the particle surface. When added to PBMC from healthy donors or from B-ALL patients, binding to 88 ± 8 % (n = 2) of all CD8+ cells in healthy, as well as samples, in which the high majority of cells wereCD8-negative. Reporter gene expression was detectable on 85 ± 5 % (n = 4) out of all CD8+ cells. On-target selectivity (percentage of CD8+ among GFP+ cells) reached 98.1 ± 0.8 % (n = 4). In vivo in humanized NSG mice, up to 40% of CD8+ T cells were hit upon a single vector injection. Selectivity for CD8+ T cells was close to absolute and liver burden was reduced 20-fold compared to unmodified AAV. These data demonstrate that capsid insertion of DARPins mediates highly selective and efficient gene transfer into mouse and human T lymphocytes. DART-AAVs have the potential to broaden in vivo gene therapy options substantially and to facilitate translatability between animal preclinical studies and human applications.