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WHO’s 2013 PMTCT guidelines recommended lifelong antiretroviral therapy (ART) for HIV-infected pregnant and breastfeeding women, exclusive breastfeeding (EBF), nevirapine prophylaxis (NVP) and early infant diagnosis (EID) for HIV-exposed-breastfed infants. We examined the association between knowledge and adherence to these guidelines among 550 HIV-infected pregnant women in Maharashtra, India. Knowledge of PMTCT guidelines was assessed using a structured-questionnaire during enrollment. Adherence to ART, NVP, and EBF was assessed using structured questionnaires during post-partum home visits at 2 weeks, 2, and 6 months respectively. EID adherence at 6 weeks was assessed by weekly infants’ HIV testing clinical record review. We used Fisher's exact test to assess the association between correct knowledge and subsequent adherence and logistic regression to estimate the odds ratios. The results showed that women’s correct responses to specific questions of each PMTCT guideline correlated with subsequent adherence. For instance, awareness of duration and place to obtain ART was associated with a higher likelihood of taking ART at delivery (aOR = 1.93, p = 0.02 and aOR = 3.91, p < 0.01 respectively). Similarly, women knowing only breastmilk should be given to infants for 6 months showed highest adherence to EBF (aOR = 2.59, p = 0.02). Women who correctly knew the reason for infant NVP administration were more adherent to it at 6 weeks (aOR = 1.77; p = 0.03). Women aware of mother-to-child HIV transmission during delivery had highest adherence to EID at 6 weeks (aOR 3.58, p = 0.01). Gaps were identified in women's detailed knowledge of and adherence to each PMTCT guideline. Suboptimal adherence to ART (n = 389, 71%), EBF (n = 179, 33%), NVP (n = 428, 78%), and EID at 6 weeks (n = 369, 67%) was found. Our study suggests providing a comprehensive knowledge of each guideline through the PMTCT program’s education strategies can enhance adherence among HIV-infected women. [ABSTRACT FROM AUTHOR]