tRNA L y s 3 is the primer for HIV-1 reverse transcriptase (RT) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNA L y s 3 , multiple forms of tRNA L y s 3 are detected in the virus released into the cell culture media. These tRNA L y s 3 isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analysis using a tRNA L y s 3 -specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNA L y s 3 to be the major abundance form of tRNA L y s 3 in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNA L y s 3 species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3' end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNA L y s 3 with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNA L y s 3 . These radioactive tRNA L y s 3 species retained their relative mobilities when rechromatograped on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNA L y s 3 . These results indicate that the major tRNA L y s 3 species present in the virus is also the major tRNA L y s 3 isoacceptor used as the primer for reverse transcription.