Background The World Health Organization's World health report 2006: Working together for health underscores the importance of human resources for health. The shortage of trained health professionals is among the main obstacles to strengthening low-income countries' health systems and to scaling up HIV/AIDS control efforts. Traditional health practitioners are increasingly depicted as key resources to HIV/AIDS prevention and care. An appropriate and effective response to the HIV/AIDS crisis requires reconsideration of the collaboration between traditional and biomedical health providers (THPs and BHPs). The aim of this paper is to explore biomedical and traditional health practitioners' experiences of and attitudes towards collaboration and to identify obstacles and potential opportunities for them to collaborate regarding care for patients with sexually transmitted infections (STIs) and HIV/AIDS. Methods We conducted a cross-sectional study in two Zambian urban sites, using structured questionnaires. We interviewed 152 biomedical health practitioners (BHPs) and 144 traditional health practitioners (THPs) who reported attending to patients with STIs and HIV/AIDS. Results The study showed a very low level of experience of collaboration, predominated by BHPs training THPs (mostly traditional birth attendants) on issues of safe delivery. Intersectoral contacts addressing STIs and HIV/AIDS care issues were less common. However, both groups of providers overwhelmingly acknowledged the potential role of THPs in the fight against HIV/AIDS. Obstacles to collaboration were identified at the policy level in terms of legislation and logistics. Lack of trust in THPs by individual BHPs was also found to inhibit collaboration. Nevertheless, as many as 40% of BHPs expressed an interest in working more closely with THPs. Conclusion There is indication that practitioners from both sectors seem willing to strengthen collaboration with each other. However, there are missed opportunities. The lack of collaborative framework integrating maternal health with STIs and HIV/AIDS care is at odds with the needed comprehensive approach to HIV/AIDS control. Also, considering the current human resources crisis in Zambia, substantial policy commitment is called for to address the legislative obstacles and the stigma reported by THPs and to provide an adequate distribution of roles between all partners, including traditional health practitioners, in the struggle against HIV/AIDS.