This study discusses the operation of a Homogeneous Charge Compression Ignition (HCCI) engine using simulated biogas and diethyl ether (DEE) as the primary and secondary fuels respectively. Performance, emission and combustion parameters are measured for various biogas flow rates (8–16 litre/min), composition (50 to 100% methane), intake temperatures (35–80 °C) and loads (20 to 90% of full load). A comparison of injecting DEE at the intake port (port injection) and 7 cm upstream (manifold injection) is also presented. It was demonstrated that at high flow rates and/or with high methane content, biogas can effectively substitute DEE and provide up to 60% of the total energy input. However, the use of lower biogas flow rates and methane fraction resulted in better thermal efficiency (as high as 26%) as well as lower HC emissions. HCCI operation offered simultaneously low NOx (< 0.2 g/kWh) and smoke (~1%). Intake heating ensured better performance and emissions, although it increased the tendency to knock. In general, manifold injection provided superior engine output compared to port injection. The knock limit of HCCI operation could be effectively extended by employing high biogas flow rates and/or methane enrichment. ANOVA study showed that biogas flow rate had a strong influence on most engine output parameters. While methane fraction correlated well with combustion indices, engine torque had a significant effect on thermal efficiency and emissions. © 2021 Elsevier Ltd