A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed μ-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120(IIIB), R5-tropic gp120(ADA), or dual-tropic gp120(MN) was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120(IIIB)-induced neuronal death; however, in combination with gp120(MN), morphine caused sustained increases in the rate of neuronal death compared to gp120(MN) alone that were prevented by naloxone. Alternatively, gp120(ADA) significantly increased the rate of neuron death, but gp120(ADA) toxicity was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120(IIIB) were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs.