After fighting his way through three three-set qualifying matches to reach the main draw, Isner, 23, beat seventh-seeded Albert Montanes of Spain 7-6 (4), 7-5.
The tall right-hander had 12 aces, put 71 percent of first serves in play and converted two of three break points to beat Montanes in 1 hour, 29 minutes.
Reynolds battled for 2 hours, 42 minutes to beat fellow qualifier Oscar Hernandez of Spain 7-6 (4), 5-7, 6-2.
Tiny margins separated the 26-year-old American and the 30-year-old Spaniard. Hernandez served 10 aces and Reynolds nine; both succeeded with 57 percent of first serves in 16 service games. But the Spanish player had eight double faults and the American only two; Reynolds converted two of 12 break points and his opponent one of four.
In other matches Tuesday, defending champion and fifth-seeded Philipp Kohlschreiber of Germany shrugged off a shoulder injury for which he needed painkilling injections to beat two-time champion Dominik Hrbaty 6-1, 6-2 in only 52 minutes.
"I didn't play the last two days but the shoulder is getting better," Kohlschreiber said. "I expected a very tough game today but I started to play very well."
Hrbaty said he was feeling the effects of a tough week in Perth where he won the Hopman Cup for Slovakia with Dominika Cibulkova.