Calcium influx through L-type voltage-gated calcium channels (VGCC) is required for extracellular-signal-regulated kinase (ERK) activation induced by gonadotropin-releasing hormone (GnRH) in pituitary gonadotropes. The current studies investigate VGCC-sensitive catalytic activities that may lie upstream of ERKs within the GnRH signaling network. Ion exchange fractionation of T3–1 cell lysates subjected to anti-phospho-tyrosine Western blot analysis revealed a nifedipine-sensitive activity that co-localized with proline-rich tyrosine kinase (Pyk) 2 immuno-reactivity. Phosphorylated Pyk2 was present in T3–1 cells following GnRH agonist administration for a time course that lasted up to four hours. Pyk2 phosphorylation was also evident in gonadotropes in vivo following administration of a bolus of GnRH. Knockdown of Pyk2 using specific siRNAs revealed Pyk2 contributed to modulation of GnRH-induced ERK but not c-Jun N-terminal kinase (JNK) activation. Using pharmacological approaches, calmodulin (Cam) was also demonstrated to be required for the phosphorylation of Pyk2. Pyk2 was shown to bind specifically to a Cam agarose affinity column in a calcium-dependent manner suggesting Cam and Pyk2 are capable of forming a complex. Specific mutation of a putative Cam binding motif within the catalytic domain of Pyk2 blocked association with Cam and uncoupled Pyk2's ability to activate ERK-dependent gene transcription. Thus, GnRH induces Pyk2 tyrosine phosphorylation dependent upon calcium flux within gonadotropes. Further, association of Pyk2 and Cam may be required to mediate the effects of calcium on Pyk2 phosphorylation and subsequent activation of ERKs by GnRH.