Filamentous viral ribonucleoproteins (RNPs) 12 to 16 nm in diameter and 100 to 1,260 nm in length, and characteristic of the genus Tenuivirus, were detected by transmission electron microscopy in purified extracts of needles collected from two mature, asymptomatic black spruce (Picea mariana) trees in New York, but not in extracts of needles from nursery seedlings. Purified RNPs from one tree had a buoyant density in CsCl = 1.39 g/cm3 and an A 260/280 = 1.436. Four ssRNA segments of 1.3, 2.1, 2.3, and 3.5 kb, but not the 8- to 9-kb fragment characteristic of most tenuiviruses, were detected in purified RNA extracts. Amplification products of the expected size were observed when RNA extracts from the two spruce trees and Maize stripe tenuivirus (MStpV), but not from tobacco, Chenopodium quinoa, or spruce seedlings were subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers to the p3 open reading frame (ORF) of MStpV vRNA 3. However, only MStpV amplified when primers to the nucleocapsid ORF (pc3 ORF on vcRNA 3) were used. Similarly, only MStpV amplified by immunocapture polymerase chain reaction (PCR) using antiserum to MStpV and primers to the p3 ORF. Sequence comparisons suggest that two distinct tenuiviruses occur in black spruce, one more closely related to MStpV than the other. One of these tenuiviruses was detected in one of 10 additional black spruce trees tested, but not in trees of six other coniferous species sampled in the Adirondack Mountains of New York.