Cellular expression of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene promotes cell death in the presence of specific nucleoside analog substrates such as acyclovir (ACV). We have reported that lymphoid CD4+ cells harboring an HSV1-TK gene, under the transcriptional control of the HIV-1 long terminal repeat (HUT-TK), are completely protected from HIV-1 spread in the presence of 10 μM ACV. In this report we clarify the efficiency, generality, and mechanism of this protective effect. We show that the protection from HIV-1 spread in HUT-TK cells obtains from both an inhibition of HIV reverse transcription by ACV metabolites and an HIV-induced and ACV-dependent cell killing. We also demonstrate that monocytic cells harboring the HIV-1-inducible HSV1-TK gene are protected from HIV spread in the presence of ACV. These observations facilitate the design of therapeutic strategies to limit HIV replication based on HSV1-TK expression.