Extra- and intracellular nucleases are predicted to decrease the in vivo efficiency of liposomal transfection. DNASE1 (D1) has been proposed as the main nuclease barrier, yet liposome-complexed DNA and in vitro lipofection are generally immune to D1. In contrast, medium conditioned by the macrophage enzyme DNASE1-like 3 (DNASE1L3 or D3) erects a potent in vitro barrier to liposomal transfection. Although homologous to D1 over its amino-terminal sequence, D3 has a distinct, highly basic carboxy terminus, which resembles polylysine stretches often found in polycationic liposomal reagents. If this domain is truncated from D3, the resulting enzyme has more nuclease activity against naked DNA ("free DNA"-nuclease activity), yet does not block transfection. C-terminal fusion of this domain to D1 forms a chimeric protein able to block transfection. D3 can be immunodetected in both serum and macrophage lysates. Macrophage-conditioned medium contains both "free DNA"-nuclease activity and the ability to block transfection, and by zymogram only a 28-kDa DNASE, consistent by size with D3, is present. Thus, medium containing D3 confers to cells an in vivo shield to the nuclear acquisition of exogenous DNA. Modulation and further elucidation of this activity are likely to have importance for both gene therapy and autoimmune disorders.