Light-emitting diode (LED) microscopy has recently been endorsed by the World Health Organization (WHO). However, it is unclear whether LED is as accurate and cost-effective as Ziehl-Neelsen (ZN) microscopy or mercury vapour fluorescence microscopy (MVFM) in tuberculosis (TB)-HIV-co-infected subjects. Direct and concentrated sputum smears from TB suspects were evaluated using combinations of LED microscopy, ZN microscopy and MVFM. Median reading time per slide was recorded and a cost analysis performed. Mycobacterial culture served as the reference standard. 647 sputum samples were obtained from 354 patients (88 (29.8%) were HIV-infected and 161 (26%) were culture-positive for Mycobacterium tuberculosis). Although overall sensitivity of LED compared with ZN microscopy or MVFM was similar, sensitivity of all three modalities was lower in HIV-infected patients. In the HIV-infected group, the sensitivity of LED microscopy was higher than ZN microscopy using samples that were not concentrated (46 versus 39%; p = 0.25), and better than MVFM using concentrated samples (56 versus 44; p = 0.5). A similar trend was seen in the CD4 count <200 cells · mL(-1) subgroup. Median (interquartile range) reading time was quicker with LED compared with ZN microscopy (1.8 (1.7-1.9) versus 2.5 (2.2-2.7) min; p ≤ 0.001). Average cost per slide read was less for LED microscopy (US$1.63) compared with ZN microscopy (US$2.10). Among HIV-TB-co-infected patients, LED microscopy was cheaper and performed as well as ZN microscopy or MVFM independent of the staining (ZN or auramine O) or processing methods used.