Fluminense showed signs of fatigue after being crowned champions last weekend, spoiling their own fans' celebrations by losing 2-0 at home to Cruzeiro.
The Rio-based side won 3-2 away at Palmeiras last weekend to become champions, but their first home game since claiming the title did not go to plan as Cruzerio took a 24th minute lead through a Walter Montillo penalty, before Jose Elber added a second shortly after half time.
The result also means Fluminense have only two more chances to get the win they need to break Sao Paulo's points record for a 38-match season. Fluminense have 76 points, two shy of the record.
Second and third-placed sides Gremio and Atletico maintained their positions with late comebacks earning them both a point.
Second-placed Gremio had to come from 2-0 down to salvage a point away to struggling Portuguesa, who led through Magalhaes Moises' 53rd minute penalty and a Leo Silva strike just after the hour mark.
Andre Lima and Jose Ze Roberto scored in the 74th and 78th minute, respectively, to earn a share of the spoils for Gremio.
Atletico Mineiro were also fortunate to get a point as Ronaldinho scored in the final minute in a 2-2 home draw with Atletico Goianiense, completing their own comeback from 2-0 down.
The visitors stunned their hosts with a fourth minute opener from Santos Raylan and then a second from Anderson Patrick after 17 minutes.
Araujo Rever got Atletico Mineiro back into it only three minutes later, but it was not until the dying seconds that they secured the point.
Fourth-placed Sao Paulo continued the theme of comebacks but theirs ended in a win as they beat Nautico Recife 2-1 at home.
Elierce Souza put the visitors ahead four minutes into the second half, but Luis Fabiano levelled six minutes later and Rogerio Ceni won it from the penalty spot.
Things were more straight forward for Corinthians in a 2-0 away win over Internacional, the goals from Paulo Guerrero and Edenilson coming in the dying seconds of each half.
That saw Corinthians leapfrog Botafogo into sixth as the latter went down 2-0 at Sport Recife.
Gilberto put the hosts ahead after 58 minutes and Henrique wrapped it up in the final minute.
Darcy Neto's late goal gave Bahia a 1-0 win over Ponte Preto, while Vagner Love scored two minutes from time to rescue a 1-1 draw for the 10 men of Flamengo against Palmeiras, who had led through a Santos Vinicius goal soon after the hour mark. Flamengo's Paulo Sergio was sent off six minutes from time.
Information from the Press Association was used in this report.