Candidate gene studies on the basis of biological hypotheses have been a practical approach to identify relevant genetic variation in complex traits such as pain, though it can be easily biased. Based on their roles in pain pathways, it is suggested that GTP cyclohydrolase, an enzyme that regulates the biosynthesis of the enzyme cofactor tetrahydobiopterin (BH4), is a modulator of peripheral neuropathic, inflammatory and even experimental pain in humans (Tegeder et al, Nature Med 2006). We have examined the effects of variations of loci in the human GTP cyclohydrolase gene (GCH1) on acutely induced experimental thermal/cold and clinical pain responses in humans. Normal subjects (443 females and 292 males) were genotyped for the 18 single nucleotide polymorphisms (SNPs) from GCH1. Haplotypes were generated from the GCH1 in 4 major ethnic populations. We evaluated normal European American subjects (194 females and 173 males) for cold and heat pain sensitivity and analyzed its association with the individual SNPs and haplotypes in the GCH1. Those requiring removal of third molars (N=177) then underwent surgical removal of 3-4 third molars and provided ratings of pain intensity every 20 minutes postoperatively for 20–240 mins (mean =122 mins) using a 10 cm visual analog scale (VAS). Unlike the report by Tegeder et al, no significant association was found between pain sensitivity and any of genetic polymorphisms including haplotypes in GCH1 in the responses to experimentally or clinically induced acute pain. The negative results between GCH1 genetic variations and pain sensitivity in our study may differ from other studies due to different populations and separate pain phenotype from different types of pain stimuli. To overcome these potential biases, whole genome association studies on pain are needed.