The molecular basis for the resistance of the sheep parasitic nematode Haemonchus contortus to the benzimidazole (BZ) group of anthelmintics was investigated. Three BZ-susceptible and three resistant populations from different geographical locations were characterized with respect to the egg-hatch assay with thiabendazole (TBZ), mebendazole (MBZ) binding tests and restriction fragment length polymorphism (RFLP) after Southern blotting. Cloned H. contortus alpha- and beta-tubulin genes were used as probes to analyze the RFLPs of genomic DNA prepared from mixtures of infectious larvae (L3) or adults. The susceptible populations showed, with both alpha- and beta-tubulin probes, 2 to 6 different fragments, depending on the restriction enzyme used. The three resistant populations showed as many fragments with the alpha-tubulin probe as the susceptible populations, but when probed with beta-tubulin only 1 or 2 fragments were visible, but always less than in the susceptible populations. An in vitro selection experiment was carried out using a susceptible population that was isolated in the laboratory before BZ came on the market. The results showed that after two selections with increasing amounts of TBZ, the population had become resistant, according to the egg-hatch assay values and MBZ binding assay. Using RFPL, the number of beta-tubulin probe reactive DNA fragments was reduced from 5 to 1. Analysis of the DNA of individual male adults of susceptible populations indicated a heterogeneity among the individual worms regarding the number of beta-tubulin probe reactive fragments (1 to 4) and frequency of the specific fragments. Usually, only one specific fragment (9 kb) was found in the resistant individuals. This 9-kb fragment was already present in some individuals in the susceptible population although it was in combination with other fragments. This would imply that genes conferring BZ resistance were present in H. contortus populations before BZ came on the market, and could explain the fast selection for BZ resistance in the field.