To study the spectrum of vaginal microflora in postmenopausal women on hormone replacement therapy (HRT) and to compare the efficacy of Papanicolaou (Pap) smears with other methods for their detection. Eighty postmenopausal women were recruited for the study. These included 40 women who had attained spontaneous and were on HRT (User 1); 20 hysterectomised women on only estrogen therapy (User 2) and 20 controls (Non users). Their clinical data was recorded and specimens were collected for vaginal cultures (for aerobic bacteria and fungi), vaginal pH, Gram stain and Pap stain on cervical-vaginal smears and toluidine blue on wet smears. Vaginal pH was significantly lower in Users as compared to Non users. Lactobacilli and Gardnerella were more frequently isolated from Users while Bacteroides and E. coli were more common in Non users. Cultures were significantly more sensitive than Gram stained direct vaginal smears in detection of aerobic bacteria; however, Candida could be detected on Gram stain alone in all the cases. Frequency of detection of organisms significantly improved by application of Gram stain to the cervico-vaginal smears. However, clinically relevant organisms like Candida, Gardnerella and Mobiluncus could be identified on Pap smears alone in >50% cases. Lactobacilli could be readily identified in Pap smears in 98% cases. Wet mounts could detect cocci more easily as compared to Pap smears. Altered vaginal microbial profile in post menopausal women receiving HRT may cause bacterial and fungal vaginitis. Although culture studies remain the gold standard to detect these microorganisms, Pap and Gram stains and wet smears provide useful supplements and may be used as alternative procedures especially in resource limited settings lacking adequate culture facilities.