Our previous work demonstrated that the type I GnRH receptor resides exclusively and constitutively within membrane rafts in T3-1 gonadotropes, and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G_q, c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRH receptor and ERK signaling. In the studies reported here, we used subcellular fractionation and co-immunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in T3-1 cells and in whole mouse pituitary. Cholesterol depletion of T3-1 cells reversibly blocked the association of both the GnRH receptor and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility following cholesterol normalization supported the conclusion that re-establishment of the association of the GnRH receptor and ERKs with the lipid raft compartment was not sufficient for reconstitution of signaling activity. In T3-1 cells, the GnRH receptor and ERK2 co-immunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRH receptor also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary, and co-immunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRH receptor to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.