A pYZ series of fission yeast expression vectors, derivatives of the pREP series, was designed to allow positive identification of cloned gene insertion and fusion to the green fluorescent protein (GFP) gene for in vivo analysis of gene expression. To validate this new vector system, the human immunodeficiency virus type 1 (HIV-1) vpr gene of viral isolate pNL4-3 was expressed in the pYZ1N vector. Vpr-induced phenotypic changes were the same as those observed with vpr expressed from pREP1N. Consistent with observations in mammalian cells, a Vpr-GFP fusion protein localizes on the nuclear membrane of fission yeast cells. Additionally, we were able to detect a naturally occurring mixture of vpr genes from a plasma sample of an HIV-infected pediatric long-term surviving patient. These pYZ vectors expedite gene cloning for general purposes and are particularly suited for largescale random gene screening.