Measurement of myocardial blood flow by 13NH3 relies heavily on the assessment of both the input function and the variable tissue extraction fraction. In six open-chest dogs, myocardial and arterial 13NH3 activity was measured both by in vitro sampling and by in vivo positron emission tomography (PET). Regional myocardial blood flow was forced to vary in the range 0.2-5 ml/min/g and actual values were assessed by in vitro counting of 153Gd microspheres. The ammonia input function was processed by: (a) total curve integration; (b) curve integration for 2 min; (c) integral of a fitted curve (gamma variate in vivo and exponential of the downslope in vitro). Method C brought to regional flow values which best approximated microspheres data. The in vitro correlation allows for correcting in vivo values for the flow-dependent extraction fraction. The method can be easily applied for regional myocardial blood flow measurements with PET in human studies.