"Again the Lord called, 'Samuel!' And Samuel got up and went to Eli and said, 'Here I am; you called me.' 'My son,' Eli said, 'I did not call; go back and lie down.' Now Samuel did not yet know the Lord: The word of the Lord had not yet been revealed to him." NIV translation
This is the second time that the Lord called to Samuel and he did not recognize the Lord. We see that Samuel did not yet know the Lord and so was being called unto salvation. This is a reminder to us that there can be people serving the Lord in the church and they can still be lost. We also see that leaders such as Eli can be blind to the working and call of God.
"A third time the Lord called, 'Samuel!' And Samuel got up and went to Eli and said, 'Here I am; you called me.' Then Eli realized that the Lord was calling the boy. So Eli told Samuel, 'Go and lie down, and if he calls you, say, "Speak Lord, for your servant is listening."' So samuel went and lay down in his place." NIV translation
The third time that this happened, Eli recognized the calling of the Lord. He told Samuel how to answer and this is a picture of how we are to share the gospel. We are to recognize when God is calling someone and tell them how to answer the call.
"The Lord came and stood there, calling as the other times, 'Samuel! Samuel!' Then Samuel said, 'Speak, for your servant is listening.' And the Lord said to Samuel: 'See, I am about to do something in Israel that will make the ears of everyone who hears about it tingle.'" NIV translation
The last two calls of the Lord to Samuel were to bring him into his ministry as a prophet. We see that the Lord is about to reveal something to him that is going to happen in Israel. We also see that there is a difference between service and ministry. Samuel had been serving for years but now, only after answering the call to salvation, is he equipped for ministry.