The effect of working fluid on the start-up and thermal performance in terms of thermal resistance and heat transfer coefficient of a pulsating heat pipe have been studied in the present paper. Methanol and de-ionized water has been selected as the working fluid. The minimum startup power for DI water was obtained at 50% filling ratio and for methanol at 40%. The optimum filling ratio in terms of minimum startup power and minimum thermal resistance was 50% for DI water and 40% for methanol. The minimum thermal resistances for DI water and methanol were observed at vertical orientation. The evaporator side heat transfer coefficient for water was slightly more, while the condenser side heat transfer coefficient was appreciably more than that of methanol. Studies were also conducted for start-up time and temperature at different orientations and it was found that the PHP charged with methanol worked efficiently at all orientations.