In a small initial study of oncogene activation in differentiated thyroid cancer, using DNA transfection and tumorigenicity assays, we identified activating ras mutations in 80% (4/5) of follicular carcinomas, but in only 20% (2/10) of papillary carcinomas (Lemoine et al., 1988). However, Suarez et al. (1988), using the 'focus' transformation assay, reported two out of a total of three papillary carcinomas to have activated ras oncogenes, while Fusco et al. (1987), also using DNA transfection techniques, failed to find any activating ras mutations in 20 papillary cases analysed. These conflicting results indicated the need for further study of the prevalence of ras mutations in thyroid cancer.We now report a further series of papillary carcinomas, analysed by polymerase chain reaction (PCR) amplification and oligonucleotide probing of archival material, and summarise our data on the incidence and type of mutation in a total of 33 differentiated thyroid carcinomas.Wax-embedded tissue blocks of eight papillary thyroid carcinomas (up to 7 years old) were obtained from the archives of the Department of Pathology, University of Wales College of Medicine. Sections of 5.m were deparaffinised twice with xylene, washed twice with ethanol and vacuum desiccated (Shibata et al., 1988). Dried pellets were mixed with sterile water, denatured for 10 min at 95°C and rapidly cooled to 4'C. PCR incubations were prepared with a Perkin Elmer/Cetus Gene Amp kit according to the manufacturers' protocol. Final concentrations were 10 mM Tris pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1 tIM of each of a pair of amplimers and 2.5 units of Taq DNA polymerase (Saiki et al., 1986). Amplimers were 20 bases long, each pair enclosing an amplified region of 100 bp. Samples received 50 cycles of amplification in a Cetus thermal cycler, with 1 min denaturation at 94°C, 2.5 min annealing at 55'C and 3 min extension at 72°C. A 10 pl aliquot of each incubation was run on a 2.5% agarose gel to check for specific amplification, and reactions were repeated until sharp single bands were obtained.After an initial quantitation, 5-12gdl of each PCR-amplified mixture was diluted to 200 t l I in 1O mM Tris pH 8.0/ 1 mM EDTA, denatured at 95°C for 5 min and cooled rapidly to 4'C. Replicate slotblots, prepared using Hybond-N filters (Amersham) and a Millipore vacuum slotblot apparatus were prehybridised at 56°C for 30 min in 3 M tetramethylammonium chloride (TEMAC)/50 mM Tris pH 7.5/2 mM EDTA/0.3% SDS/5 x Denhardts solution, 100 flg ml-' denatured herring sperm DNA (Wood et al., 1985). Filters were hybridised for 1-2 h at 56°C with 32P-endlabelled 20mer oligonucleotide probes specific for mutations altering aminoacids at the 12, 13, and 61 positions of each of the three human ras genes. As a check on quantitation, filters were first autoradiographed after non-stringent washes in 2 x SSPE/0.1% SDS at room temperature. They were then Correspondence: