Background: Ruxolitinib, an orally bioavailable inhibitor of the JAK family of kinases, may play a role in the treatment of childhood cancers with JAK1, JAK2, CRLF2, or other BCR-ABL1-like (Ph-like) alterations. We performed a phase 1 and pharmacokinetic (PK) study of ruxolitinib in children with relapsed/refractory solid tumors (STs) or hematologic malignancies (HMs; leukemias or myeloproliferative neoplasms). Methods: Ruxolitinib was administered orally twice daily (BID) continuously in 28 day cycles. The starting dose (15 mg/m²; dose level 1, DL1) was equivalent to the recommended adult dose of 25 mg/dose BID. Using the rolling six design, subsequent 20% dose escalations were 21 (DL2), 29 (DL3), 39 (DL4), and 50 (DL5) mg/m²/dose. Patients with HMs were enrolled at one dose level below ST patients. PK and pharmacodynamic analyses of phosphorylated (p) JAK2, STAT5, and S6 were performed in Cycle 1. Results: Twenty-eight ST and 21 HM patients were enrolled (median age 14.4 years; range 2.4 - 21.4). Twenty-seven ST and 10 HM patients were evaluable for toxicity. Ruxolitinib was generally well-tolerated, although there was 1 grade 5 event at DL2 in a ST patient (multi-system organ failure). During the dose escalation phase in STs, there was 1 dose-limiting toxicity at DL3 (neutropenia), DL4 (neutropenia), and DL5 (increased CPK). Grades 3 and 4 non-dose-limiting adverse events related to therapy in STs included cytopenias, nausea, and elevated transaminases and creatinine. PK parameters were similar to those in adults with marked inter-patient variability in the C_max and AUC. Median peak plasma inhibitory activity of pJAK2 (44.8%), pSTAT5 (58.9%), and pS6 (62.3%) appeared generally dose-independent with the exception of greater pSTAT5 inhibition at DL5. Conclusions: The recommended phase 2 dose of ruxolitinib is 50 mg/m² orally BID. A trial of ruxolitinib with chemotherapy for children with acute lymphoblastic leukemia harboring activating JAK mutations or fusions or other Ph-like alterations is in development. Clinical trial information: NCT01164163.