Unacylated ghrelin (UAG) accounts for approximately 80% of circulating ghrelin, but does not bind to or activate the receptor for acylated ghrelin (AG), GHS-R1a (1). However, UAG exerts a distinct spectrum of activity, promoting adipogenesis in bone marrow (2), but not abdominal fat (WAT; 3) and inhibiting skeletal growth (4). In the absence of an alternative receptor, we investigated the potential role of GHS-R in mediating these skeletal effects of UAG. As previously shown in rats (2), intra-bone marrow (ibm) infusion of AG and UAG (from an osmotic minipump connected to a tibial ibm catheter implanted under isoflurane anaesthesia) induced adipogenesis in male wild-type (WT) mice, increasing the number of tibial marrow adipocytes from 22±3 cells/field (vehicle-infused; n=5) to 42±1 cells/field (AG; n=5; P<0.001) and 37±3 cells/field (UAG; n=4; P <0.05; all data mean±SEM, compared by 1-way ANOVA and Bonferroni post hoc test). Surprisingly, this effect was abolished in male loxTB-GHS-R (GHS-R-null) littermates, AG- and UAG-treated mice having 100 (n=5) and 88% (n=5) of the adipocytes in vehicle-treated GHS-R-null animals (n=5). Neither AG nor UAG had any effect on tibial epiphyseal plate width (EPW). Immunocytochemistry revealed that, unlike stomach (which expressed the activating enzyme, ghrelin O-acyl transferase (GOAT), but not the adipocyte-specific marker, PPARγ) and intra-abdominal WAT (which expressed PPARγ, but not GOAT), rat tibial marrow adipocytes co-expressed both PPARγ and GOAT. Furthermore, the adipogenic effect of a tibial ibm UAG infusion (as above) was abolished in male GOAT-null mice (41±2 cells/field in vehicle-treated (n=6) vs 48±3 cells/field in UAG-treated (n=6)), compared to a 120% increase in UAG-treated male WT littermates (16±3 in vehicle-treated vs 35±6 in UAG-treated; n=5,6; P <0.05). Again, UAG had no effect on tibial EPW. However, both GHS-R-null and GOAT-null mice displayed elevated marrow adiposity compared to their WT littermates (P <0.01), suggesting that elevated circulating UAG may produce maximal adipogenesis in these mice. ELISA of terminal plasma samples revealed that circulating ghrelin (total) was not elevated in either GHS-R-null or GOAT-null mice. Thus, our data demonstrate that the adipogenic effect of UAG in bone marrow is dependent upon acylation by GOAT and activation of GHS-R. The ability of cells expressing these proteins to activate UAG prior to signalling through GHS-R represents a novel endocrine mechanism. Indeed, the cell-specific distribution of these proteins in conjunction with the differential uptake mechanisms for AG and UAG may thereby determine the unique activity spectra of these two hormones.