Introduction: Many in vitro studies have debated over the appropriate apical preparation size and taper for the irrigant to reach the apical third of root canals; however little is known about irrigant penetration in vivo. Aim: The purpose of this in vivo study was to determine the effect of apical preparation size and taper using two different needles on the irrigant penetration into the apical part of root canals. Materials and Methods: A 120 human mandibular teeth indicated for root canal treatment was selected for the study. The study was done in two phases. The first phase aimed to determine the smaller apical size that will allow irrigant penetration to working length. Disto buccal canal of with mandibular 1st molar was selected for study and was sequentially instrumented to 30/0.04 and 35/0.04. All patients were randomly divided into two groups for irrigation: Group A (n=30) 30G flat open ended needle and Group B (n=30) 30G side vented needle. The second phase of the study aimed to determine the taper that will allow irrigant penetration to working length. Teeth were instrumented to 30/0.04 and 30/0.06 and for irrigation patients were randomly divided into group A and B (n=30). Diatrizoate sodium contrast medium was used to measure irrigant penetration with digital radiograph. A blinded observer measured the distance between the working length and maximum irrigant penetration. Results: In both groups A and B, increasing the preparation size of the DB canal from 30 to 35 increased irrigant penetration which was statistically insignificant (p>0.05). But irrigant penetration in 6% taper of DB canal was significantly better than 4% (p< 0.001) for both the groups. Flat open ended needles performed significantly better than side vented needles in both phase I and II of the study (p< 0.001). Conclusion: Within the limitations of the study it can be concluded that an apical preparation size of 30 and 6% preparation taper in DB canal of mandibular molars allow adequate irrigation penetration in the apical third with flat open ended needles performing better than side vented needles.