Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4− cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2–6], epithelial cells [5,7], CD4− lymphocytes and thymocytes [8,9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4− cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4+ cells. This process requires contact between the coreceptor-bearing and CD4+ cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4− cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.