Aspects of glutamine metabolism were examined in two related hybridomas, a high-producing line (PQX B1/2) and a low-producing line (PQX B2/2). The growth and metabolic characteristics of both cell lines were identical or very similar. During batch growth glutamine was completely exhausted from the medium and an examination of the fate of [C-14]glutamine highlighted the importance of this amino acid as an energy source. The relative enzyme activities and the amount of ammonia produced during growth indicated that glutamine is oxidized preferentially via the transmination pathway. The overall rate of glutamine utilization from the growth medium was similar to the rate of [C-14]glutamine uptake which suggests that transport may regulate glutamine metabolism.