An epidemiological investigation indicated that six patients treated in a haematology unit who developed acute hepatitis B may have been infected as a result of contamination of a liquid nitrogen bone marrow storage tank. The clinical details are described elsewhere (Tedder et al., 1995); we describe the virological methods used to support the findings. HBV DNA was amplified from sera using a nested PCR with primers for the surface gene, and a region encompassing precore, the 3' end of X, and the 5' end of core. HBV DNA was also extracted from the frozen detritus in the liquid nitrogen storage tank. After equilibration, the aqueous material was filtered, co-precipitated with albumin and polyethylene glycol and the HBV DNA extracted by phenol-chloroform and ethanol precipitation. Direct nucleotide sequence analysis indicated that four patients were infected with HBsAg subtype adw viruses which carried novel amino acid substitutions at codons 145 and 146 of the X gene. HBV DNA extracted from the storage tank detritus contained identical sequences. The samples from two other patients, subtype ayw, did not contain the novel sequence changes in X and had other sequence differences. These findings linked conclusively the four patients as a cluster and the rescue of HBV-DNA sequences from the contaminated storage tank by the method described confirmed this as the common source of infection. Two other HBsAg-positive patients were excluded from the cluster by sequence analysis. Demonstration of infection by this route has implications for the safe storage of bone marrow and other related biological materials.