Many conclusions concerning cell culture mycoplasmas are based on data from studies in fibroblast cultures. Some conclusions may not be valid in other types of differentiated cell cultures. M. salivarium was isolated from 35 human lymphocyte cultures (HLC), 34 from the same laboratory. The organism grew to more than 10(8) colony forming units (CFU) per ml of lymphocyte suspensions and was readily detectable by microbiological culture, uridine phosphorylase, and uridine/uracil assays. Direct mycoplasmal assays on HLC by DNA fluorochrome staining and scanning electron microscopy (SEM) yielded artifacts that interfered with diagnosis. For DNA and SEM of HLC, inoculation into indicator cell cultures is recommended. M. salivarium infection of HLC did not produce any immediate difference in growth rates; however, infected cultures eventually died 14 to 29 passages after infection in contrast to uninfected controls. The same organism in 3T6 fibroblasts effected a 60% decrease in growth rate. Although M. salivarium is a frequent isolate from the oral cavity, it is a rare cell culture isolate. M. salivarium was able to initiate growth over a wide pH range, grew as well in cell cultures as in cell-free media, and was resistant to 50 microgram per ml of gentamycin, tylocine, kanamycin, and erythromycin. By Cot1/2 analysis, M. salivarium had a genomic molecular weight of 4.2 X 10(6) daltons. M. salivarium did not increase chromosome aberrations in one HLC. Some of these results have application to infection of HLC by other mycoplasmal species.