Topically delivered pain medications can provide adequate pain relief without the side-effects of systemically delivered drugs. This open-label, pharmacokinetic study in 36 healthy adults was designed to determine plasma levels of a topically delivered cream containing 4% amitriptyline and 2% ketamine (NP-1). The level of quantitation was (0.5 ng/mL). 4 mL of NP-1 cream were applied b.i.d. during a 48 hour period. Safety/tolerability were assessed and blood samples were collected up to 96 hours after first application. Plasma amitriptyline levels were above 0.500 ng/mL after the second application, through 60 hours after the last application. The mean Cmax of 1.676 ng/mL occurred 72 hours after the first application. The highest amitriptyline concentration observed at any one time point was 5.91 ng/mL. The terminal elimination phase for amitriptyline had not been reached in most of the subjects. Plasma ketamine levels were above 0.500 ng/mL within several hours after the first application up to the last observation at 96 hours. The highest single ketamine concentration was 10.7 ng/mL. The mean ketamine Cmax of 5.198 ng/mL occurred at 41.4 hours. Terminal elimination was ongoing during the final 48 h of the study in the majority of the subjects. The PK parameter values for both drugs were age-group independent. NP-1 was found to be safe and well tolerated across all groups. Mild, transient application site irritation was the most common adverse event and occurred in 11% of subjects. Prior studies utilizing a twice-daily local application of NP-1 in the treatment of various neuropathies suggest that significant pain relief can be achieved. This study confirms that the efficacy is due to topical action as only sub-therapeutic blood levels of both drugs are observed.