In skim milk obtained from susceptible cows after intramammary and intravenous inoculation (primary infected milk), foot-and-mouth disease (FMD) virus type O1 was slower inactivated by heat treatment than virus that had been added to pre-exposure skim milk. Residual virus infectivity in heated primary infected milk was more efficiently detected in bovine thyroid cell cultures than in secondary pig kidney (PK2) cell cultures. Untreated primary infected milk was found to inhibit both FMD-virus and vesicular stomatitis virus plaque formation in PK2 cells, suggesting the presence of interferon. The results of further tests confirmed that the interfering activity in unheated primary infected milk was indeed caused by an interferon. Interferon excretion in primary infected milk was investigated using a series of milk samples from three cows. Maximum interferon titres were found after 24 h, coinciding with or shortly after the first virus excretion peak. The results are discussed with particular reference to the use of primary infected milk in studies of the thermal inactivation of FMD-virus.