OBJECTIVES: Despite good evidence that isoniazid preventive therapy (IPT) reduces incidence of tuberculosis among people with HIV infection, implementation of IPT is low. This study aimed to describe barriers to IPT implementation from healthcare provider and patient perspectives in a donor-funded HIV care programme in Gauteng province, South Africa, in which IPT is recommended, but delivery is variable. DESIGN: A qualitative study using in-depth interviews and a focus group discussion. METHODS: We conducted interviews with 22 clinic staff and 20 patients from 10 purposively selected HIV clinics, and a staff focus group discussion. Staff were questioned on their knowledge and experience of IPT, and asked about barriers to its use. Patients were asked for their opinions about taking IPT. RESULTS: Healthcare workers reported the primary barrier to IPT use was lack of knowledge and experience. Prescribers were unaware of the benefits of IPT and unclear about guidelines. The belief that existing screening tools are inaccurate in HIV-infected individuals and the need to refer patients to separate clinics for tuberculosis screening also emerged as barriers. No patients had heard of IPT. CONCLUSION: Barriers to the widespread use of IPT primarily derived from healthcare workers, in particular, lack of experience among physicians. In addition to overcoming operational barriers, a change in healthcare worker perception is needed if IPT is to be widely used; we suggest local clinical opinion leaders could help achieve this.