Objective: Determine if varying ultrasound frequency affects the delivery of 10% hydrocortisone concentrations during phonophoresis. Utilize intramuscular microdialysis probe for drug collection, thus improving the experimental model. Methods: Thirty one (10 in groups 1 and 2, 11 in group 3) healthy subjects participated in this study. Interventions: Subjects were randomly assigned to one of three treatment groups receiving 10 minute ultrasound treatments applied to a standardized area of the gastrocnemius muscle of the right leg. The ultrasound was performed over the treated area using a 10% hydrocortisone compound mixed with standard ultrasound gel. The contralateral limb served as the control (no mixed compound or treatment) for all groups. Group one received sham ultrasound. Medicated gel was placed on the treatment site, the sound head moved, but no ultrasound was applied. Group two received 45 KHz at .056 w/cm2. Group three received 1 MHz at 1.0 w/cm2 at a 50 % duty cycle. Results: There was no difference in cortisol concentration change during treatment between the three treatment groups on the treated limbs (sham = 1.1 ±7.5 ng/ml, 45 KHz = 1.1 ± 1.5 ng/ml, 1 MHz = 4.1 ± 7.8 ng/ml; F2,22 = .34, P = .72) or control limbs (sham = 1.65 ± 6.6 ng/ml, 45 KHz = -1.3 ± 2.7 ng/ml, 1 MHz = 0.37 ± 8.1 ng/ml; F2,22 = .67, P = .546). No difference was found in cortisol concentration change during treatment between the treatment limbs and the control limbs (treatment = 2.1 ± 6.2 ng/ml, control = 0.20 ± 5.9 ng/ml; F1,22 = .9, P = .35). The following factors were found to influence cortisol concentrations levels in dialysate collected during treatment: depth of muscle in the treatment limbs (F1,22 = 6.4, P = .02), microdialysis probe depth in the control limbs (F1,22 = 4.1, P = .05), and pre treatment cortisol level in the control limbs (F1,22 = 10.1, P = .004. Conclusions: There was no evidence altering ultrasound frequency from 45 KHz to 1 MHZ enhanced the delivery of 10% hydrocortisone to treatment tissues under these experimental conditions.