The authors conducted a study to determine the validity of two commercially available in-office dental unit waterline test kits compared to the gold standard, R2A agar. Samples were collected from the air/water syringes of dental units and cultured on HPC Samplers, Petrifilm AC Plates, and R2A agar plates. HPC Samplers and R2A agar plates were incubated for 7 days and counted manually using magnification. Petrifilm AC Plates were counted after incubation time of 5 and 7 days using an electronic-plate reader. Validity measurements were calculated using a cutoff value < or = 500 colony-forming units per milliliter. The accuracy for the HPC Sampler compared to R2A agar was 71%. The accuracy for the Petrifilm AC Plates at 5 and 7 days was 79% and 87% compared to R2A agar. The Petrifilm AC Plate (7-day incubation) demonstrated higher sensitivity, specificity, predictive values, and accuracy than the HPC Sampler kit.