To investigate the clinical validity of newer diagnostic tests such as monitoring of EBVqPCR and lymphocyte function assay ImmuKnow in helping to diagnose PTLD in pediatric lung transplant recipients. Single-center, retrospective case-control study. CsA trough levels, EBVqPCR and ImmuKnow (Cyclex Inc., Columbia, MD, USA) levels were measured serially as part of routine care. Re-transplant patients and patients who did not reach 12 months post-transplant at the time of analysis were excluded. Twenty-seven patients met the inclusion criteria. The study group consisted of seven patients who developed PTLD, five of which were EBV- recipients who received EBV+ lungs. The rest of the eligible patients served as controls. Median time to develop PTLD was 273 days (range: 166-343). One, two, three, six, and nine months after transplant, mean (+/-s.d.) CsA trough whole blood levels (ng/mL) were not different between the two groups: 378 +/- 38, 390 +/- 52, 402 +/- 89, 359 +/- 42, and 342 +/- 115, vs. 416 +/- 105, 347 +/- 64, 337 +/- 78, 333 +/- 86, and 281 +/- 54 [PTLD vs. no-PTLD, respectively (p > 0.05 for all time points)]. Mean (+/-s.d.) EBVqPCR levels (copies/mL) measured at three, six, and nine months post-transplant were significantly elevated in PTLD group compared to no-PTLD group: 84 +/- 99, 3384 +/- 7428 and 839 +/- 1444 vs. 9 +/- 26, 8 +/- 36 and 32 +/- 136, respectively (p < 0.05 for all time points). Mean (+/-s.d.) ImmuKnow levels (ATP ng/mL) at three, six, and nine months post-transplant were significantly lower in the PTLD group when compared with no-PTLD group: 144 +/- 67, 137 +/- 110, and 120 +/- 153 vs. 290 +/- 161, 300 +/- 162, and 293 +/- 190, respectively (p < 0.05 for all time points). Close monitoring of EBV viral load by qPCR and the degree of immunosuppression via ImmuKnow may guide physicians to reach the diagnosis of PTLD early.