The level of transgene expression following intravenous administration of AAV2 vectors to mice varies with sex, with males expressing 5- to 13-fold higher levels than females at the same dose of vector (Davidoff et al, Blood 2003, 102:480-488). This effect is independent of the AAV capsid, the promoter/transgene expression cassette and is postulated to be associated with androgen-enhanced transition from single stranded to double stranded DNA following gene transfer. We have extended this concept by testing the hypothesis that there is a difference in serum |[alpha]|1AT levels between male and female mice following intrapleural gene transfer with AAVrh.10, a highly effective AAV derived from rhesus macaque. Male or female C57Bl/6 mice were injected by intrapleural or intravenous route with 5|[times]|1010 gc of AAVrh.10|[alpha]|1AT and serum |[alpha]|1AT levels were assessed at 0, 4, 14 and 28 days. Consistent with our prior studies in males only using the intravenous route (De et al, Mol Ther 2004, 9:S128), the serum |[alpha]|1AT level at 28 days was 1520|[plusmn]|380 |[mu]|g/ml in male mice. Strikingly, however, the |[alpha]|1AT levels at 28 days were 300|[plusmn]|89 |[mu]|g/ml in females (p<0.05), a 5.1-fold difference. Sex differences were also observed with intrapleural administration: the serum |[alpha]|1AT level at 28 days was 1360|[plusmn]|250 |[mu]|g/ml in males and 480|[plusmn]|58 |[mu]|g/ml in females, a 2.8-fold difference (p<0.05 male vs female). By both routes, human |[alpha]|1AT was found in serum 4 days following gene transfer; and even at that time point, the serum |[alpha]|1AT levels were higher in males than in females (p<0.05). The sex difference in |[alpha]|1AT levels following intrapleural administration of AAVrh.10|[alpha]|1AT may be due to leakage of vector to the systemic circulation and thereby to liver where males expressed higher |[alpha]|1AT levels. Therefore, an AAVrh.10 vector expressing a non-secreted transgene, luciferase, was injected intrapleurally into male and female mice and the luciferase activity of various organs assessed after 1 wk. As expected, there was a 4.0-fold higher luciferase activity in the livers of male mice. But there was also a striking sex difference in other organs with diaphragm and chest wall, the major sites of transgene expression following intrapleural administration, showing a 6.5- and 4.0-fold higher expression level respectively in males. Similarly, the left lung showed a 9.6-fold higher expression level in males. By contrast two organs where luciferase levels were low, the quadriceps and kidney showed no significant difference in expression levels between males and females. We conclude that preferential expression of transgene from AAV vectors in male mice is not confined to intravenous administration and may occur in multiple organs. Although the responsible mechanism is not entirely clear, it is striking how an AAVrh.10 vector that rapidly yields over 100 times the expression level that is derived from AAV2 vectors shows a similar pattern of sex-specific transgene expression. The data suggest that sex is a significant parameter that needs to be considered in both toxicology studies and human clinical trials of AAV derived vectors.