Mechanisms of intracellular survival of M. paratuberculosis (M. ptb) in macrophages are poorly understood and have been difficult to study for lack of precise methods to quantify viable M. ptb. Conventional plate counting gives low viable counts due to clumping of the organism and plates often become contaminated or dehydrated during the prolonged incubation required. Microscopic counts of infected macrophages on coverslips are tedious, time consuming, affected by clumping and can not distinguish viable from nonviable M. ptb. We previously described a radiometric (BACTEC) method for quantification of slow growing mycobacteria (Appl. Env. Microbiol. 54:910,1988). In the present study, we demonstrate application of this technique to the study of ingestion and intracellular killing of M. ptb by bovine monocytes and monocyte-derived macrophages (Mo). Freshly isolated monocytes were infected with M. ptb (10:1 organism to cell ratio). After 2 h, and 4, 8, 12, and 16 days of infection, the monolayers were lysed with SDS. The lysates were inoculated into BACTEC 12B vials supplemented with mycobactin. Growth was monitored every two days on a BACTEC 460 and cumulative growth index (CGI) readings were calculated. CGI values were translated into viable M. ptb counts on the basis of a standard curve. Results were obtained in 20 days and at less cost in labor and materials than for other counting methods. M. ptb counts by microscopic examination of stained coverslips of infected Mo monolayers and by BACTEC quantification of M. ptb from Mo lysates were compared. Over 90% of maximal M. ptb uptake by Mo occurred within 20 min. The effects of cytokines and several other factors affecting the number of viable M. ptb numbers of Mo will be reported.