At the dawn of human genetics Sir Archibald Garrod used alkaptonuria as a paradigm to demonstrate the applicability of the Mendelian laws to men and to develop the concept of inborn errors of metabolism. The human cDNA for homogentisate 1,2 dioxygenase was identified due to its homology to the corresponding mouse enzyme and was screened for mutations in alkaptonuric patients from Slovakia. Homozygous mutations were found in four unrelated families and their segregation with the disease was demonstrated. One of the mutations, observed in two families, leads to a frame-shift and thus is unlikely to produce functional protein. The data formally establish the homogentisate 1,2 dioxygenase gene (HGD) as the molecular cause of alkaptonuria and allow for the development of molecular carrier tests in populations at risk.