There are limited data on adherence to HIV treatment guidelines. We assessed adherence to US Department of Health and Human Services guidelines with Australian Commentary for adults initiating antiretroviral therapy (ART). Data were recorded regarding "when to start", "what to start" and pre-ART comorbid disease assessment for consecutive adults initiating ART at primary care and hospital clinics in Sydney and Melbourne from 2004 through 2008. Independent predictors of adherence to guidelines were calculated by stepwise logistic regression. For the 500 subjects (95.9% male, mean 40.2 years, median CD4 count 270 cells/μL) "when to start" adherence was 87.6%, and was less likely with initiation in a clinical trial [0.25 (95% CI: 0.13 to 0.49); P < 0.0001] and previous, short-term nontherapeutic antiretroviral exposure [0.08 (0.03 to 0.25); P < 0.0001]. "What to start" adherence was 69.0% for guideline-"preferred" regimens (85.8% for guideline-"preferred" or "alternative" regimens) and more likely with ART initiated in 2008 versus pre-2008 [OR: 2.69 (1.64 to 4.61); P = 0.0001]. Median comorbid disease assessment adherence was 56.8%, ranging from 25.6% for urinalysis to 99.2% for white blood cell count, and was more likely in patients with AIDS, and initiating ART in hospital or in a clinical trial. Hospital clinics were more likely to perform antiretroviral resistance testing (71.2% vs. 46.4%, P < 0.0001), to use "preferred" ART regimens (76.8% vs. 62.2%, P = 0.0002) but less likely to promote healthy diet and lifestyle (63.4% vs. 36.4%, P < 0.0001). "When to start" and "what to start" guidelines have been largely adhered to in Australia, but pre-ART comorbid disease assessment requires greater attention.