Cancer metastasis to the skeleton is the cause of significant morbity and mortality in prostate cancer patients. Despite its prevalence, the mechanisms involved in the development of bone metastases remain unclear. A metastatic initiating cell (MIC) phenotype CD44+/CD24- has been identified in prostate cancer, but a complementary approach to aid MIC cell recognition, involves detection of aldehyde dehydrogenase (ALDH) activity. This study characterises the proliferative and invasive functional behaviour of prostate cancer (PC-3) cell ALDH subpopulations in vitro. We also used a new in vivo model developed by our laboratory, to study bone micrometastasis and interaction with the bone marrow endothelial cells. A metatarsal from a newborn mouse (1-3 day old) was engrafted into a Dorsal Skinfold Chamber (DSC) implanted on a severe combined immunodeficient (SCID) mouse (5-6 weeks old) under systemic ketamine/xylazine anaesthesia. PC3 labelled with red fluorescent protein (RFP) cells were sorted into ALDHhigh (7%) and ALDHlow (20%) cell-subpopulations using the ALDEFLUORkit and flow cytometry. The subpopulations (1×105 cells) were administered via intra-cardiac injection into groups of animals (n=10). An additional group was used to evaluate the homing response of ALDHhigh cells treated with weekly zoledronic acid for 4 weeks (zol; intraperitoneal, 25µg/kg/injection; 100μg/kg total). Microscopy recordings of the chamber tissue and bone-graft were made at 48hr intervals for up to 4 weeks. At the end of the study, tissue was harvested and processed for microCT, multi-photon analysis, histology and immunohistochemistry. ALDH subpopulation cell proliferation, migration and invasion were investigated via functional in vitro assays. ALDHhigh cells home to bone and interact with the bone microvascular endothelium in significantly higher numbers than ALDHlow cells (Day 15: mean ± SEM; ALDHhigh 12.3±1.8 cells vs. ALDHlow 1.9±0.5 cells; p<0.05). Treatment with zol does not influence ALDHhigh PC3 cells homing to bone and intereacting with the microvascular endothelium and bone microenvironment in vivo. PC3-ALDHhigh cells display increased proliferation, migration and invasion ability in vitro. In summary PC3-ALDHhigh exhibit functional characteristics associated with enhanced metastatic potential in vitro, with increased homing to and interaction with the bone microvasculature and metastatic-niche in vivo, which appear resistant to a standard current treatment regime.