Ten crows (Corvus brachyrhynchos) and three blue jays (Cyanocitta cristata), species indigenous to North America, were intravenously inoculated with 10(3) PFU of West Nile virus (WNV) strain NY99 for production of positive tissues for Canadian surveillance. Both species developed clinical signs 4 days postinoculation (dpi). Virus was detected in blood, cloacal and tracheal swabs, and in a number of organs by reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation (titers reaching over 10(7) PFU/0.1 g). Virus appeared as early as 1 dpi in blood (10(2)-10(3) PFU/ml) and spleen (10(3)-10(4) PFU/0.1 g of tissue), whereas kidney, liver, intestine, gonads, heart, skeletal muscle, and lung tested positive for WNV in a later stage of the infection. Immunostaining (IHC) using heterologous rabbit anti-WNV polyclonal antiserum detected viral antigen in a wide range of organs, starting at 2 dpi. Detection of WNV antigen in the brain of blue jays and crows by IHC was laborious as only few cells, not present in all sections, would stain positive. Mononuclear cells appeared to be an important target for virus replication, contributing to virus spread throughout tissues during the infection. This conclusion was based on the positive IHC staining of these cells in organs before virus antigen detection in parenchymal cells and supported by virus isolation and RT-PCR-positive results in white blood cells. The inability of blue jays and crows to perch and fly may reflect weakness due to generalized infection and marked skeletal muscle involvement, although involvement of the central nervous system cannot be excluded.